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Four new Ophiostoma species associated with conifer- and hardwood-infesting bark and ambrosia beetles from the Czech Republic and Poland

Fungi under the order Ophiostomatales (Ascomycota) are known to associate with various species of bark beetles (Coleoptera: Curculionidae: Scolytinae). In addition this group of fungi contains many taxa that can impart blue-stain on sapwood and some are important tree pathogens. A recent survey that focussed on the diversity of the Ophiostomatales in the forest ecosystems of the Czech Republic and Poland uncovered four putative new species. Phylogenetic analyses of four gene regions (ITS1-5.8S-ITS2 region, ß-tubulin, calmodulin, and translation elongation factor 1-α) indicated that these four species are members of the genus Ophiostoma. All four newly described species can be distinguished from each other and from closely related species based on DNA sequence comparisons, morphological characters, growth rates, and their insect associations. Based on this study four new taxa can be circumscribed and the following names are provided: Ophiostoma pityokteinis sp. nov., Ophiostoma rufum sp. nov., Ophiostoma solheimii sp. nov., and Ophiostoma taphrorychi sp. nov. O. rufum sp. nov. is a member of the Ophiostoma piceae species complex, while O. pityokteinis sp. nov. resides in a discrete lineage within Ophiostoma s. stricto. O. taphrorychi sp. nov. together with O. distortum formed a well-supported clade in Ophiostoma s. stricto close to O. pityokteinis sp. nov. O. solheimii sp. nov. groups within a currently undefined lineage A, which also includes Ophiostoma grandicarpum and Ophiostoma microsporum. This study highlights the need for more intensive surveys that should include additional countries of Central Europe, insect vectors and host tree species in order to elucidate Ophiostoma species diversity in this region.


Introduction
The order Ophiostomatales includes seven well supported lineages represented by the following genera: Aureovirgo, Ceratocystiopsis, Fragosphaeria, Graphilbum, Hawksworthiomyces, Raffaelea s. stricto, and Sporothrix. Two additional major groups, for which monophyly is not well supported, are Leptographium s. lato and Ophiostoma s. lato (De Beer and Wingfield 2013;De Beer et al. 2016). The Ophiostomatales also contain some smaller lineages with uncertain taxonomic positions, such as lineages A, B, C and D .
Species of Ophiostoma Syd. & P. Syd. (Sydow and Sydow 1919) reside in Ophiostoma s. stricto (Ophiostomatales, Ascomycota) (De   (De Beer and Wingfield 2013;Linnakoski et al. 2016;Yin et al. 2016;Chang et al. 2019). The genus Ophiostoma currently includes nearly 40 described taxa, most of which are associated with phloem and wood-dwelling beetles. The most important morphological features that can be used to describe these fungi are ascomata with short to long necks, crescent to allantoid shaped ascospores, and pesotum-, hyalorhinocladiella-or sporothrix-like asexual morphs (De Beer and Wingfield 2013). Most Ophiostoma species produce spores in sticky droplets that can easily attach to the exoskeletons of their insect vectors (Malloch and Blackwell 1993). The genus Ophiostoma includes plant-associated species with varying degrees of pathogenicity. Most members are considered as nonpathogenic, especially in their endemic range, where they have co-evolved with their host tree species, and are mainly responsible for causing blue-stain in freshly exposed sapwood (Wingfield et al. 2017). However, some Ophiostoma species are highly virulent tree pathogens that have been responsible for tree death in natural as well as managed forest ecosystems (Harrington 1993). In many cases, pathogenicity and tree damage caused by these fungi are linked to their introduction into new areas (Loo 2009;Wingfield et al. 2015).
Members of the Ophiostomatales, that exist in symbiosis with bark beetles in Central Europe, have been mainly described from Austria (e.g. Kirisits 2001), Germany (e.g. Kirschner 2001), Poland (e.g. Siemaszko 1939;Jankowiak 2005Jankowiak , 2006Jankowiak , 2008Jankowiak and Bilański 2013) and from a limited number of reports from the Czech Republic and Slovakia (e.g. Kotýnková-Sychrová 1966). These studies reported numerous species belonging to the Ophiostomatales that were in association with conifer-and hardwoodinfesting bark beetles. However, the diversity of ophiostomatoid fungi associated with Abies alba, Larix decidua and hardwood trees are not well studied. For this reason, several comprehensive studies have been undertaken in recent years to explore the diversity of ophiostomatoid fungi in Central Europe (Jankowiak et al. 2017a(Jankowiak et al. , 2019. As part of a fungal diversity survey conducted in the Czech Republic and Poland (Jankowiak et al. 2017a, 2019) a total of 30 undescribed Ophiostomatales taxa associated with hardwood-and conifer-infesting beetles were uncovered. Until now, only six of these have been formally described as new species (Jankowiak et al. 2017bAas et al. 2018).
In this study, both morphological characters and DNA sequence data from the ITS region (ITS1-5.8S-ITS2), and three protein coding genes (b-tubulin, calmodulin, translation elongation factor 1-a) were analysed to a) characterise the four new species within Ophiostoma s. lato, and compare them to closely related known species within the Ophiostomatales, and b) to provide a formal description for these new species.

Materials and methods
Isolates and herbarium specimens Bark beetles and galleries were collected during a study conducted by Jankowiak et al. (2017aJankowiak et al. ( , 2019. Fungal isolations were made from beetles collected in Poland, and this included the following beetle species: Pityokteines vorontzowi, Pityokteines curvidens, Anisandrus dispar, Taphrorychus bicolor and Scolytus intricatus (Fig. 1). In addition, materials investigated in this study included fungi that were isolated from Ips cembrae in the Czech Republic (Table 1). Fungal isolation strategy and the origin of some of the isolates used in this study have been described previously (Jankowiak et al. 2017a(Jankowiak et al. , 2019. All fungal isolates used in this study are listed in Table 1. The isolates are maintained in the culture collection of the Department of Forest Pathology, Mycology and Tree Physiology; University of Agriculture in Krakow, Poland. The ex-type isolates of the new species described in this study were deposited in the Westerdijk Fungal Biodiversity Institute (CBS), Utrecht, the Netherlands, and in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa. Herbarium specimens have been deposited in the Herbarium of the University of Turku (TUR), Finland. Taxonomic descriptions and nomenclatural data have been registered in MycoBank (www. MycoBank.org) (Robert et al. 2013).

DNA extraction, PCR and sequencing
The fungal isolates were grown on 2% malt extract agar [MEA: 20 g Bacto TM malt extract (Becton-Dickinson and Company, Franklin Lakes, USA), 20 g agar (Bacto TM agar powder from Becton-Dickinson and Company, Franklin Lakes, USA), 1 l deionized water] in 90 mm plastic Petri dishes for 1-2 weeks prior to DNA extraction. DNA was extracted using the Genomic Mini AX Plant Kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer's protocol.
Amplified products were sequenced with the BigDye Ò Terminator v 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the products were resolved with a ABI PRISM 3100 Genetic Analyzer (Applied Biosystems), at the DNA Research Centre (Poznań, Poland) using the same primers that were used for the PCR. The sequences (Table 1) were compared with sequences retrieved from GenBank using the BLASTn algorithm (Altschul et al. 1990). Newly obtained sequences were deposited in NCBI GenBank (Table 1).

Phylogenetic analyses
BLAST searches using the BLASTn algorithm were performed to retrieve similar sequences from Gen-Bank (http://www.ncbi.nlm.nih.gov) and accession numbers for these sequences are presented in the corresponding phylogenetic trees (Figs. 2 and 3, S1-S4). Datasets were curated with the Molecular Evolutionary Genetic Analysis (MEGA) v6.06 program (Tamura et al. 2013). The ITS dataset included all available sequences for reference species in  Ophiostoma s. lato that could be retrieved from Gen-Bank ( Fig. 2) to show the placement of our isolates within this genus. The outgroup taxon for the ITS dataset analysis was Sporothrix abietina and S. stenoceras. The three protein coding gene regions were sequenced for 29 (bT, and TEF1-a) and 25 (CAL) of our isolates (Table 1). Datasets were analysed individually and with regards to the protein coding sequences as concatenated constructs. Sequence alignments were performed using the online version of MAFFT v7 (Katoh and Standley 2013 (Hall 1999), and for the protein coding regions the alignments were compared with gene maps previously published by Aas et al. (2018) to ensure that introns and exons were aligned appropriately. The resulting alignments and trees were deposited into TreeBASE (TB2:S24036). 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 (AICc) 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/). The ML analysis included bootstrap analysis (1000 bootstrap pseudoreplicates) in order to assess node support values and the overall reliability of the tree topology.
MP analyses were performed with PAUP* 4.0b10 (Swofford 2003). Gaps were treated as fifth state characters. Bootstrap analysis (1000 bootstrap replicates) was conducted to determine the levels of confidence for the nodes within the inferred tree topologies. Tree bisection and reconnection (TBR) was 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 analysed 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). The four MCMC chains were run for 10 million generations applying the best-fit model. Trees were sampled every 100 generations, resulting in 100,000 trees. The Tracer v1.4.1 program (Rambaut and Drummond 2007) was utilized to determine the burn-in value for each dataset. The remaining trees were utilized to generate a 50% majority rule consensus tree, which allowed for calculating posterior probability values for the nodes.
Morphology, growth studies and mating tests Morphological characters were examined for selected isolates and for the herbarium specimens chosen to represent the type specimens for the newly proposed species. Cultures were grown on 2% MEA with or without host tree twigs to induce potential ascocarp formation. Autoclaved twigs with bark were positioned in the centre of the MEA agar plates. Fungal cultures were derived from single spores, and crossings were made following the technique described by Grobbelaar et al. (2010). To encourage the production of ascomata for species descriptions, single conidial isolates were crossed in all possible combinations. Cultures were incubated at 25°C and monitored regularly for the appearance of fruiting structures.
Morphological features were examined by mounting materials in 80% lactic acid on glass slides, and observing various fruiting structures using a Nikon Eclipse 50i microscope (Nikon Ò Corporation, Tokyo, Japan) with an Invenio 5S digital camera (DeltaPix Ò , Maalov, Denmark) to capture photographic images. Microscopy was done as previously described by Kamgan Nkuekam et al. (2011). Colour designations were based on the charts of Kornerup and Wanscher (1978).
For each taxonomically relevant structure fifty measurements were made, whenever possible, with the Coolview 1.6.0 software (Precoptic Ò , Warsaw, Poland). Averages, ranges and standard deviations were calculated for the measurements, and these are presented in the format '(min-)(mean -SD)-(mean ? SD)(-max)'.
Growth characteristics for the four newly proposed species were determined by analysing the radial growth for four isolates in pure culture that represent each of the studied species (Table 1). Agar disks (5 mm diam.) were cut from actively growing margins of fungal colonies for each of the tested isolates and these disks were placed in the centre of plates containing 2% MEA. Four replicate plates for each of the isolates studied were incubated at 5, 10, 15, 20, 25, 30 and 35°C. Colony diameters (two measurements per plate) were determined 7 d after inoculation and radial growth rates were calculated as mm/d.

Morphological characteristics
The four new taxa showed differences with regards to growth rates in culture and colour differences ranging from rust brown, grey brown, to olive brown (Table 2). Taxon 1 and Taxon 2 produced abundant synnemata that were arranged either singly or in groups topped with cream-white mucilaginous spore drops. A sporothrix-like synanamorph was also present in cultures of Taxon 1. In addition, Taxon 3 and  allantoid: (2.9-)3.7-5.1 (-6.3) 9 (0.8-)1.1-1.7 (-2.2); ellipsoidal: (3.1-)3.9-4.8 (-5.3) 9 (0.9-) 1. Taxon 4 produced hyalorhinocladiella-like asexual morphs. A sexual state was induced in Taxon 3 and 4. Sexual states were not observed for Taxon 1 and Taxon 2 in any of the crosses done between different isolates. Morphological differences among these new taxa are listed in Table 2, and discussed in the Notes under the new species descriptions in the Taxonomy section.
The optimal growth temperature for Taxon 3 was at 20°C, and at 25°C for Taxon 1, 2 and 4. For all isolates the growth rate was minimal at 5°C except for Taxon 4 which failed to grow at this temperature. No growth was observed at 30°C for Taxon 1 and 3 and all Taxa failed to grow at 35°C except for Taxon 4 (Fig. 4).
The best evolutionary substitution model for ITS, bT, CAL, TEF1-a datasets was GTR ? G. Except for the TEF1-a for lineage A dataset, for which the best model was GTR ? I. The best evolutionary substitution model for the combined ITS, bT, CAL, TEF1-a datasets was GTR ? I?G. The burn-in values in BI analyses for all data matrices were 25% of the trees.
The ITS tree shows the placement of the Czech and Polish isolates (referred to as Taxon 1 to Taxon 4) within the Ophiostomatales (Fig. 2). Taxa 1-3 resided among sequences representing species that are members of Ophiostoma s. stricto, while Taxon 4 is grouped with other species in the lineage A (Fig. 2) ). Taxa 1-3 appear to group closely with members of the O. piceae species complex. Taxon 1 grouped within the O. piceae species complex, while Taxa 2 and 3 formed two adjacent lineages peripheral to the O. piceae species complex (Fig. 2). The lineage that includes Taxon 3 includes the ex-type isolate of O. distortum (Fig. 2). Strains of Taxon 1 had ITS sequences that were identical with ITS sequences noted in most members of the O. piceae species complex. Taxon 4 grouped among members of the lineage A, which includes O. grandicarpum and O. microsporum (Fig. 2). This taxon had unique ITS sequences compared with O. grandicarpum and O. microsporum.
The BI, MP, ML phylogenetic analyses of the aligned protein-coding datasets (bT, CAL, TEF1-a and combined) for members of the O. piceae species complex yielded trees with different topologies (Fig. 3, Figs. S1-S3). In the bT, CAL and TEF1-a trees (Figs. S1-S3), Taxa 1-3 formed well-supported lineages that clearly separated these four newly proposed species from all the other known species in the O. piceae species complex and other closely related species. The only exception was Taxon 3, which had differences in the bT sequence compared to the O. distortum bT sequence, but the node lacked statistical support (Fig. S1). However, the combined analyses of the bT, CAL and TEF1-a datasets clearly distinguish Taxa 1-3 into separate lineages within Ophiostoma s. stricto (Fig. 3). Analyses of the bT, CAL and TEF1-a data grouped isolates of Taxon  formed a well-supported lineage that is clearly distinct from O. macrosporum and O. grandicarpum (Fig. S4).

Taxonomy
The morphological characterization and phylogenetic comparisons based on four genetic loci, showed that four taxa associated with bark beetles from the Czech Republic and Poland (Taxa 1 to 4) are distinct from each other and from other known taxa in Ophiostoma s. lato and, therefore, are described here as new species. They are described as follows: Taxon 1 Ophiostoma rufum R. Jankowiak & P. Bilański, sp. nov. (Figure 5) MycoBank: MB830195. Etymology: The epithet rufum, referring to the rust brown colony on MEA.
Culture characteristics Colonies with optimal growth at 25°C on 2% MEA with radial growth rate 2.2 (± 0.1) mm/d, no growth occurred at 30 and 35°C. Colonies brownish orange to a rust brown, with smooth margins (Fig. 5i). Reverse rust brown. Hyphae pale yellow to olive yellow in colour (Kornerup and Wanscher 1978), smooth, submerged in the medium and aerial mycelium abundant, not constricted at the septa, 0.6-5.3 (mean 1.8 ± 1.3) lm diam. Notes This species is most closely related to O. breviusculum (Chung et al. 2006) based on the phylogenetic analyses of the ITS, and bT sequences (Figs. 2, S1). However, the DNA sequences of bT and TEF1-a (Figs. S1, S2) are unique and clearly suggested that O. rufum is distinct from O. breviusculum, and other species of the O. piceae species complex.
Morphologically, O. breviusculum can be distinguished from this new species by having shorter synnemata. Ophiostoma rufum differs from O. breviusculum by the presence of crystal-like structures in the upper part of the stipe. In addition, O. breviusculum forms brown to dark brown colonies, while the new taxon has colonies displaying brownish orange to rust brown colours.
Ophiostoma rufum was infrequently isolated from L. decidua in association with Ips cembrae in Czech Republic (Jankowiak et al. 2017a).
Culture characteristics Optimal growth temperature on MEA is 25°C with radial growth rate of 3.9 (± 0.1) mm/d, no growth occurred at 35°C. Colonies on MEA hyaline at first, later becoming coffee to dark brown in colour, floccose, with abundant grey aerial mycelium, margin smooth (Fig. 6i). Reverse dark brown. Hyphae light to dark brown in colour (Kornerup and Wanscher 1978), smooth, often fused, submerged in the medium and aerial mycelium abundant, not constricted at the septa, 0.8-5.4 (mean 1.8 ± 1) lm diam. Notes This species forms a lineage within Ophiostoma s. stricto and can be distinguished from all its (i) fourteen-day-old culture on MEA. Scale bars: a = 250 lm, b = 250 lm, c = 50 lm, d = 100 lm, e = 100 lm, f = 50 lm, g = 10 lm, h = 10 lm members by the ITS (Fig. 2) and protein coding sequences (Figs. S1-S3).
Morphologically, O. pityokteinis is most similar to members of the O. piceae species complex. In contrast to the species in the O. piceae species complex, which produce column-like synnemata, O. pityokteinis produces cup-or club-like synnemata. In addition, this species has no sporothrix-like asexual state, which is a characteristic for many species of the O. piceae species complex.
Ophiostoma pityokteinis has very specific host and vector ranges. It was found abundantly on A. alba in associations with fir-infesting bark beetles, especially Pityokteines species (Jankowiak et al. 2017a).
Culture characteristics Colonies with optimal growth at 20°C on 2% MEA with a radial growth rate at 4.2 (± 0.1) mm/d; growth at 15°C was better when compared to growth at 25°C, and no growth occurred at 30 and 35°C. Colonies brown, margins smooth (Fig. 7l). Reverse dark brown. Hyphae olive yellow in colour (Kornerup and Wanscher 1978), smooth, submerged in the medium and aerial light greyish mycelium sparse, not constricted at the septa, 0.9-4.9 (mean 2.3 ± 1.1) lm diam. Notes This species is most closely related to O. distortum (Davidson, 1971). However, the DNA sequences of ITS, bT, CAL and TEF1-a (Figs. 2, S1-S3) clearly suggested that O. taphrorychi is distinct from O. distortum.
Ophiostoma taphrorychi morphologically resembles O. torulosum, which was described from T. domesticum on F. sylvatica in Germany (Butin and Zimmermann 1972). However, it can be distinguished from O. torulosum by smaller ascospores, and the presence of a different asexual state. Ophiostoma torulosum produces sporothrix-like asexual state, while the new species has a hyalorhinocladiella-like morph producing larger primary and in some instances secondary conidia. In addition, O. taphrorychi was isolated only from T. bicolor (Jankowiak et al. 2019), suggesting a specific association with this bark beetle species. O. rufum sp. nov. was assigned to the O. piceae species complex, as defined by Harrington et al. (2001) based on ITS sequence analysis of ten hardwood and conifer-inhabiting synnematous species. The monophyly of this group in Harrington's studies (2001) was not statistically supported. However, in subsequent studies, 'hardwood' species formed a separate lineage with substantial support, a lineage that was subsequently referred to as O. quercus species complex (Kamgan Nkuekam et al. 2011) or the O. ulmi species complex (De Beer and Wingfield 2013). The conifer-inhabiting species previously included in the O. piceae species complex (Harrington et al. 2001;Linnakoski et al. 2010) did not form a monophyletic lineage in recent reports based on ITS and LSU analyses (De Beer and Wingfield 2013;Yin et al. 2016). The monophyly of the O. piceae species complex was also not well supported in the present study. Nevertheless, based on individual proteincoding genes, as well as the phylogenetic analysis of the concatenated dataset these species formed a monophyletic lineage with substantial support. Yin et al. (2016) recommended the designation of a newly defined O. piceae species complex. They also noted that members of the O. piceae species complex have similar morphological characteristics such as unsheathed, allantoid ascospores, and pesotum-like synnemata and sporothrix-like asexual morphs. The monophyly of the O. piceae species complex based on three protein-coding gene regions, including bT, CAL and TEF1-a sequence data was confirmed in the present study and O. rufum fits well into this species complex as a conifer-inhabiting species forming pesotum-and sporothrix-like asexual morphs.
O. rufum is highly similar to O. breviusculum, which was originally described from Ips subelongatus and Dryocoetes baikalicus infesting Japanese larch (Larix kaempferi) in Japan (Chung et al. 2006). The colony morphology on MEA is the main morphological difference between O. rufum and O. breviusculum. In addition, O. rufum produces shorter synnemata compared to O. breviusculum, and has unique crystalline structures in the upper part of the stipe. Ophiostoma breviusculum is considered heterothallic (Chung et al. 2006). Although we were not able to observe the sexual state, one would infer that O. rufum is also heterothallic. Ophiostoma rufum and O. breviusculum are also quite similar based on their host range and beetle vectors. Both species appear to be associated with Larix species (O. rufum with L. decidua, while O. breviusculum with L. kaempferi), and Ips species (O. rufum with I. cembrae, while O. breviusculum with I. subelongatus). However, the DNA sequences of bT and TEF1-a obtained in this study clearly suggested that O. rufum is distinct from O. breviusculum.
The present study shows that O. pityokteinis sp. nov. has a unique ITS sequence. It forms a lineage within Ophiostoma s. stricto, grouping close to the O. piceae species complex and the O. distortum lineage. This new species is characterized by cup-or club-like synnemata, and the lack of a sporothrix-like anamorph. Sexual states were not observed for this species in crosses done between different isolates, suggesting that this species could be heterothallic. O. pityokteinis also has unique ecological characteristics; this fungus appears to be commonly associated with bark beetles infesting A. alba. In our previous study (Jankowiak et al. 2017a (Yin et al. 2016). However, there are some morphological and ecological differences, mainly between the asexual states. No asexual morph is known for O. arduennense (Carlier et al. 2006), while O. torulosum and O. distortum produce sporothrix-like morphs that differ in their conidial size and shape (Butin and Zimmermann 1972;Davidson 1971). Ecologically, O. arduennense and O. torulosum have been found in association with ambrosia beetles infesting F. sylvatica (Butin and Zimmermann 1972;Carlier et al. 2006), while O. distortum was described from Pityokteines sparsus infesting various conifer trees and unknown ambrosia beetle species (Davidson 1971). Morphologically, O. taphrorychi should be compared to O. torulosum, and to a lesser degree with O. arduennense. Ophiostoma torulosum can be distinguished from O. taphrorychi by forming ascomata in concentric rings, larger ascospores, and shorter ostiolar hyphae. In addition, O. torulosum produces a sporothrix-like asexual state; while O. taphrorychi has a hyalorhinocladiella-like morph producing larger primary or secondary conidia. Ophiostoma taphrorychi (previously referred as Ophiostoma sp. 8) appears to be closely associated with T. bicolor on F. sylvatica (Jankowiak et al. 2019). This species resides together with O. distortum in a discrete, wellsupported lineage in Ophiostoma s. stricto.
In the present study, O. solheimii sp. nov. together with O. grandicarpum and O. microsporum resided in a well-supported phylogenetic group referred to as lineage A by De Beer et al. (2016) supporting the view that this lineage probably represents a distinct genus in the Ophiostomatales (De Beer and Wingfield 2013;De Beer et al. 2016). Species in this lineage have small, orange-section shaped ascospores without noticeable sheath, and ascomata with very long necks without ostiolar hyphae (sometimes necks form cup-shaped to funnel-like openings at the apex). In addition, these fungi have hyalorhinocladiella-like asexual morphs (Davidson 1942;Kowalski and Butin 1989).
O. solheimii is morphologically most similar to O. grandicarpum (Kowalski and Butin 1989). These two fungi have homothallic mating systems and perithecia with long necks, however O. grandicarpum forms substantially larger ascomatal bases (up to 950 lm in diam) and longer perithecial necks (up to 10 000 lm) compared to O. solheimii. In addition, O. solheimii has smaller ascospores and cup-shaped to funnel-like openings at the apex of the perithecial necks. Both species produce hyalorhinocladiella-like asexual morphs. However, O. solheimii has smaller conidia than O. grandicarpum. In addition, O. solheimii can also be distinguished from O. grandicarpum by colony characteristics. The new species has olive brown colonies, while O. grandicarpum forms white to cream coloured colonies. Both species inhabit similar ecological niches. O. grandicarpum is known to occur mainly on Q. robur in Poland, the Czech Republic, Germany and Russia (Kehr and Wulf 1993;Kowalski and Butin 1989;Kowalski 1991;Novotný and Š růtka 2004;Selochnik et al. 2015). The fungus was only rarely isolated from A. dispar and Scolytus intricatus on Q. robur in our previous study (Jankowiak et al. 2019). O. solheimii (previously referred as Ophiostoma sp. 9) so far has been found only in association with A. dispar on Q. robur (Jankowiak et al. 2019).
Recent surveys of conifer and hardwood-infesting bark beetles conducted in Czech Republic and Poland revealed many potentially new fungal species and new beetle-fungus associations (Jankowiak et al. 2017a(Jankowiak et al. , 2019. These findings clearly show that the Ophiostomatales associated with bark and wooddwelling beetles in Central Europe are very diverse and still poorly understood. In this study, we described four new taxa, which support the view that the diversity of these fungi is likely much higher than currently appreciated. Due to the economic impact of the Ophiostomatales it is important to formally describe species of Ophiostomatales that still remain undescribed. This will allow for a better understanding of the taxonomic status and diversity of these economically and ecologically important fungi.