Two new species of Ophiostomatales (Sordariomycetes) associated with the bark beetle Dryocoetes alni from Poland

Abstract Bark beetles belonging to the genus Dryocoetes (Coleoptera, Curculionidae, Scolytinae) are known vectors of fungi, such as the pathogenic species Grosmannia dryocoetidis involved in alpine fir (Abies lasiocarpa) mortality. Associations between hardwood-infesting Dryocoetes species and fungi in Europe have received very little research attention. Ectosymbiotic fungi residing in Ceratocystiopsis and Leptographium (Ophiostomatales, Sordariomycetes, Ascomycota) were commonly detected in previous surveys of the Dryocoetes alni-associated mycobiome in Poland. The aim of this study was to accurately identify these isolates and to provide descriptions of the new species. The identification was conducted based on morphology and DNA sequence data for six loci (ITS1-5.8S, ITS2-28S, ACT, CAL, TUB2, and TEF1-α). This revealed two new species, described here as Ceratocystiopsis synnematasp. nov. and Leptographium alneumsp. nov. The host trees for the new species included Alnus incana and Populus tremula. Ceratocystiopsis synnemata can be distinguished from its closely related species, C. pallidobrunnea, based on conidia morphology and conidiophores that aggregate in loosely arranged synnemata. Leptographium alneum is closely related to Grosmannia crassivaginata and differs from this species in having a larger ascomatal neck, and the presence of larger club-shaped cells.


introduction
Bark beetles in the genus Dryocoetes (Coleoptera: Curculionidae: Scolytinae) are mainly secondary pests infesting dead, injured, and felled or windthrown conifer-and hardwood hosts. For this reason, most members of Dryocoetes have no or only minor economic importance, although Dryocoetes confusus, the most destructive species in the genus, may cause extensive mortality of subalpine fir (Abies lasiocarpa) in North America (Bright 1963;Negrón and Popp 2009). The biology of hardwood-infesting Dryocoetes species is poorly understood in Poland. One of them is Dryocoetes alni (Georg), which has a wide geographical distribution, extending from France in the west to Siberia in the east, and from Fennoscandia in the north to Italy and Asia Minor in the south (Dodelin 2010). In Poland it occurs rarely, but probably it is widespread. This beetle species attacks weakened or dead trees of Alnus spp., Populus spp. and Corylus avellana (Gutowski and Jaroszewicz 2001;Borowski et al. 2012).
Dryocoetes beetles live in close association with fungi; most notably with members of the Ophiostomatales (Ascomycota, Sordariomycetes) that are well-recognized associates of bark-and wood-dwelling beetles (Kirisits 2004;Wingfield et al. 2017). According to Six (2012), associations among bark beetles and fungi range from mutualistic to commensal, and from facultative to obligate. Some fungi are highly specific and associated only with a single beetle species, while others can be associated with many beetle species. The majority of fungi vectored by Dryocoetes cause sapstain but some are responsible for serious tree diseases, such as the pathogenic species of Leptographium, Grosmannia dryocoetidis which is involved in A. lasiocarpa mortality (Molnar 1965). Ophiostomatales is comprised of two families, Kathistaceae and Ophiostomataceae, the latter comprising several several phylogenetic lineages that include, among others, Ceratocystiopsis, Graphilbum, Leptographium, Ophiostoma, Raffaelea and Sporothrix (Hyde et al. 2020). Members of these lineages have similar morphological and ecological characteristics. These fungi are also referred to as so-called ophiostomatoid fungi, a polyphyletic grouping characterized by the production of sticky spore masses at the apices of the flaskshaped sexual fruiting structures and their association with different arthropods (De Beer and Wingfield 2013;De Beer et al. 2013a, b, 2016Hyde et al. 2020).
Leptographium sensu lato is a broadly defined polyphyletic group of morphologically similar species (De Beer and Wingfield 2013). To date, Leptographium sensu lato includes ten species complexes and some smaller lineages with uncertain taxonomic positions (De Beer and Wingfield 2013;Jankowiak et al. 2017). The genus Leptographium contains more than 150 described taxa, most of which are associated with phloem-and wood breeding beetles that affect a wide range of plants worldwide (Jacobs and Wingfield 2001). Leptographium species colonizing the roots of conifers may cause tree health problems, such as members of the Leptographium wageneri species complex that are responsible for black stain root disease (BSRD) on conifers in western North America (Goheen and Cobb 1978). Morphologically, species of Leptographium sensu lato are characterized by mononematous, darkly pigmented conidiophores terminating in penicillate branches. In addition, species belonging to the Grosmannia olivacea species complex also form synnematous conidiophores. Some members of Leptographium sensu lato produce sporothrix-like or hyalorhinocladiella-like synanamorphs. Many Leptographium sensu lato also form sexual morphs characterized by globose ascomata with elongated necks (Jacobs and Wingfield 2001) and these were often included in the genus Grosmannia Goid. (Goidànich 1936).
Leptographium species have historically been classified into various genera including Grosmannia, Ceratocystis Ellis and Halst. (Upadhyay 1981), and Ophiostoma Syd. and P. Syd. (Seifert et al. 1993). Phylogenetic analyses based on the ribosomal large subunit (LSU) and beta-tubulin sequence data carried out by Zipfel et al. (2006) documented distinct differences between Ophiostoma and Grosmannia, and redefined the latter genus to include all Leptographium with sexual morphs. De Beer and Wingfield (2013) re-evaluated the taxonomy of Leptographium and Grosmannia, considering all available DNA sequence data for all species. The authors concluded that sequence data for additional gene regions would be necessary to fully resolve the delineation of Leptographium and Grosmannia. De Beer and Wingfield (2013) suggested that all known Leptographium and Grosmannia placed in Leptographium sensu lato based on phylogenetic inference, should be treated in their current genera (Leptographium or Grosmannia). However, new species, excluding those residing in the G. penicillata species complex, should provisionally be treated in Leptographium, irrespective of their sexual or asexual morphs.
In contrast to species of Leptographium sensu lato, members of Ceratocystiopsis are less widespread globally. The genus Ceratocystiopsis currently includes nearly 20 taxa, most of which are collected from plants infested by phloem and wood-breeding beetles. Ceratocystiopsis species have short-necked perithecia, elongated ascospores, and hyalorhinocladiella-like asexual morphs (Upadhyay 1981;De Beer and Wingfield 2013).
Surveys of hardwood-infesting bark beetles in Poland have recently led to the recovery of an unknown Leptographium species from Dryocoetes alni (Jankowiak et al. 2019a). In addition, several isolates resembling Ceratocystiopsis have also been isolated from D. alni in association with Populus tremula L. In this study, all known Leptographium and Ceratocystiopsis species as well as the newly collected isolates were compared based on morphology and DNA sequence data for six nuclear loci, with the overall aim of providing accurate identifications for these fungi.

Isolates and herbarium specimens
Isolations were made from the bark beetle D. alni and its galleries established in P. tremula logs. Strains were collected in beech-alder stand in southern Poland (Paprocice: 50°48'56.10"N, 21°2'51.23"E) during March-September 2018. The isolation procedures were the same as described by Jankowiak et al. (2019a). Isolates were also collected from Alnus incana (L.) Moench and P. tremula infested by D. alni and from Malus sylvestris (L.) Mill. infested by Scolytus mali (Bechstein) during studies conducted by Jankowiak et al. (2019a).
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

DNA extraction, PCR and sequencing
DNA extractions were done as described by Jankowiak et al. (2018). For sequencing and phylogenetic analyses, six loci were amplified: internal transcribed spacer 1 and 2 (ITS1-5.8S-ITS2), internal transcribed spacer 2 and large subunit (ITS2-28S), actin (ACT), beta-tubulin (TUB2), calmodulin (CAL) and the translation elongation factor 1-alpha (TEF1-α) using the primers listed in Table 2. DNA fragments were amplified in a 25 µL reaction mixture containing 0.25 µL of Phusion High-Fidelity DNA polymerase (Finnzymes, Espoo, Finland), 5 µL Phusion HF buffer (5x), 0.5 µL of dNTPs (10 mM), 0.75 µL DMSO (100%) and 0.5 µL of each primer (25 µM). Amplification reactions were performed in the LabCycler Gradient thermocycler (Sensoquest Biomedical Electronics GmbH, Germany). Amplification of the various loci was performed under the following conditions: a denaturation step at 98 °C for 30 s was followed by 35 cycles of 5 s at 98 °C, 10 s at 52-64 °C (depending on the primer melting temperature and fungal species) and 30 s at 72 °C, and a final elongation step at 72 °C for 8 min. The PCR products were visualized under UV light on a 2% agarose gel stained with Midori Green DNA Stain (Nippon Genetic Europe).
Amplified products were sequenced with the BigDye Terminator v 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the products were re-  (Jacobs et al. 2004) Leptographium solved with an 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 and added to previous alignments for the ITS1-5.8S-ITS2-28S and ITS2-28S regions (Jankowiak et al. 2019a). Alignments were adjusted to accommodate the new sequences and the data sets were used to obtain consensus sequences and the two data sets were concatenated.

Phylogenetic analyses
BLAST searches (Altschul et al. 1990) using the BLASTn algorithm were performed to retrieve similar sequences from GenBank (http://www.ncbi.nlm.nih.gov) and accession numbers for these sequences are presented in the corresponding phylogenetic trees (Figs 1-4). Datasets were curated with the Molecular Evolutionary Genetic Analysis (MEGA) v6.06 program (Tamura et al. 2013). The phylogenetic position of Taxon 1 was determined from their concatenated ITS1-5.8S-ITS2-28S sequences within a dataset that covered all ITS1-5.8S and ITS2-28S sequences of Ceratocystiopsis available in GenBank, as well as sequences of C. pallidobrunnea obtained in this study (Fig. 1). The outgroup taxa for the ITS1-5.8S-ITS2-28S dataset analysis were Ophiostoma karelicum and O. quercus. The TUB2 dataset included all available sequences for reference species in Ceratocystiopsis that could be retrieved from GenBank and six of our isolates (Fig. 2) in order to identify isolates to the species level. In the case of Taxon 2, the ITS2-28S dataset included most of the available sequences for reference species in Leptographium sensu lato that could be retrieved from GenBank ( Fig. 3) to show the placement of our isolates within this group. The outgroup taxa for the ITS2-28S dataset analysis was O. karelicum and O. novo-ulmi. The concatenated constructs of sequences for multiple loci (ITS1-5.8S-ITS2-28S + TUB2 + TEF1-α + ACT) were also used for 11 of our isolates, Grosmannia crassivaginata, and L. piriforme (Fig. 4). Before individual data sets for the ITS2-28S, ACT, TUB2, and the TEF1-α gene regions were used for 11 of our isolates, Grosmannia crassivaginata, and L. piriforme. The outgroup taxa for the ITS1-5.8S-ITS2-28S + TUB2 + TEF1-α + ACT datasets analysis were Leptographium flavum and L. vulnerum. Datasets concerning the protein coding sequences were concatenated. Sequence alignments were performed using the online version of MAFFT v7 (Katoh and Standley 2013). The ITS1-5.8S, ITS2-28S, ACT, TUB2, and TEF1-α 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). The resulting alignments and trees were deposited into TreeBASE (http://purl. org/phylo/treebase/phylows/study/TB2:S25615). Aligned data sets of the protein-coding genes were compared to gene maps constructed by Yin et al. (2015) to determine the presence or absence of introns and to confirm that introns and exons were appropriately aligned (Suppl. material 1: Tables S1-S3). Single nucleotide polymorphisms (SNPs) for different gene regions between the new taxa and the phylogenetically closest related species were also identified by comparative sequence analysis.
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 jMod-elTest 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. 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 for each data set. 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 agar [MEA: 20 g Bacto malt extract (Becton Dickinson and Company, Franklin Lakes, USA), 20 g agar (Bacto agar powder from Becton Dickinson and Company, Franklin Lakes, USA), 1 l deionized water] 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 Corporation, Tokyo, Japan) with an Invenio 5S digital camera (DeltaPix, Maalov, Denmark) to capture photographic images. Microscopy was done as previously described by Kamgan Nkuekam et al. (2011). Color 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, 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 two newly proposed species and Grosmannia crassivaginata (isolate CBS 119144) were determined by analyzing the radial growth for five 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 center of plates containing 2% MEA. Four replicate plates for each of the proposed new species and G. crassivaginata were incubated at 5, 10, 15, 20, 25, 30 and 35 °C. The radial growth (two measurements per plate) were determined 7 d (Taxon 1) and 4 d (Taxon 2, and G. crassivaginata) after inoculation, and growth rates were calculated as mm/d.

Morphological characteristics
The two new taxa showed differences with regards to growth rates in culture and color differences ranging from white (Taxon 1) to brownish gray (Taxon 2). Taxon 1 produced a hyalorhinocladiella-like asexual morph with simple and highly branched conidiophores, which often aggregate in loosely synnemata that were arranged either singly or in groups topped with white mucilaginous spore drops. Taxon 2 produced short mononematous conidiophores with allantoid conidia, and stalked club-shaped cells. A sexual morph could be induced in all isolates of Taxon 2; the most distinct features observed in both the herbarium specimens and the studied isolates were the short ascomatal necks and falcate ascospores with gelatinous sheaths. Sexual morph was not observed for Taxon 1 in any of the crosses done between different isolates. Morphological differences among these new taxa and the most closely related species are listed in Tables 3, 4, and discussed in the Notes under the new species descriptions in the Taxonomy section.
The optimal growth temperatures were 25 °C for Taxon 1 and 30 °C for Taxon 2. No growth was observed at 5 °C for Taxon 2.
These results indicate that the six isolates of Taxon 1 within Ceratocystiopsis and the 11 isolates of Taxon 2 within Leptographium represent novel species.

Discussion
This study identified two new species of ophiostomatoid fungi associated with Dryocoetes alni on Alnus incana and Populus tremula in hardwood ecosystems in Poland. DNA sequence comparisons and morphological features supported these as novel. The species were named Ceratocystiopsis synnemata and Leptographium alneum. The results confirm earlier findings that many species of the Ophiostomatales are associated with hardwoodinfesting bark beetles in Poland (Jankowiak et al. 2017(Jankowiak et al. , 2019aAas et al. 2018).
The results of this study revealed one new species of Ceratocystiopsis bringing the total number of species in the genus to 17. The newly described species is morphologically similar to other species of Ceratocystiopsis, with hyalorhinocladiella-like asexual morph (De Beer et al. 2013a). In contrast to other Ceratocystiopsis species, C. synnemata produces simple as well as highly branched conidiophores reminiscent of C. pallidobrunnea (Olchowecki and Reid 1974;Upadhyay 1981) or C. rollhanseniana (Hausner et al. 2003). In addition, C. synnemata forms synnemata with loosely packed conidiophores that appear to be a unique feature of Ceratocystiopsis.
Ascomata in Ceratocystiopsis tended to be globose with short necks, and falcate ascospores surrounded by a gelatinous sheath (De Beer and Wingfield 2013). Generally, Ceratocystiopsis species produce perithecia in varying degrees of abundance and maturity (Upadhyay 1981;Plattner et al. 2009). Ceratocystiopsis synnemata did not form a sexual state in crosses between different isolates. That would suggest that this species is heterothallic or produces perithecia very sparsely.
Most of the formally described species of Ceratocystiopsis are known only from Pinaceae including those in the genera Picea, Pinus and Pseudotsuga. For example, in Poland, two species of Ceratocystiopsis have previously been reported: C. minuta and species of uncertain status, C. alba, which both have been isolated from spruceinfesting bark beetles (Jankowiak et al. 2009). Only C. pallidobrunnea was collected from hardwood tree species, Populus tremuloides in Canada (Olchowecki and Reid 1974;Plattner et al. 2009). The discovery of C. synnemata on Populus tremula in this study clearly show that Ceratocystiopsis species are also distributed in hardwood forest ecosystems in Europe.
A new species of Leptographium was discovered from Dryocoetes alni in this study. This new taxon is closely related to Leptographium piriforme and Grosmannia crassivaginata forming a well-supported lineage distinct from other species of Leptographium sensu lato. All these three species have the curved conidia formed on short conidiophores, club-shaped cells, short-necked perithecia, and falcate, sheathed ascospores. These features clearly distinguish them from the other species recognized in the various species complexes currently recognized within Leptographium sensu lato.
Based on DNA sequence comparisons, L. alneum described in this study is closely related to G. crassivaginata, a species described from Picea mariana, Populus grandidentata and P. tremuloides in Canada (Griffin 1968). Morphologically, L. alneum most closely resembles G. crassivaginata. The asexual morph of L. alneum produced short conidiophores, and stalked club-shaped cells, similar to those of G. crassivaginata. Other similarities are the presence of short-necked perithecia, and fusiform-falcate, sheathed ascospores. Leptographium alneum, however, differs from G. crassivaginata, in having longer ascomatal neck and larger club-shaped cells. Moreover, L. alneum has larger conidia than G. crassivaginata (CBS 119144), especially with regard to the width of the conidia. Other differences are the presence of barrel-shaped or globose cells that make up the conidiophores, which in G. crassivaginata are occasionally only slightly swollen. There are also differences in characteristics of cultures. Leptographium alneum has brownish gray cultures in contrast to olive brown culture of G. crassivaginata (isolate CBS 119144). Both species belong to fast-growing species on MEA, however L. alneum grows much faster than G. crassivaginata, especially at 35 °C.
Leptographium species are generally considered as saprotrophs or pathogens of conifer trees (Jacobs and Wingfield 2001). However, the results of the present study confirm previous Polish investigations that some of the Leptographium species have a close affinity to hardwoods in Europe. Recently, L. betulae, L. tardum, and L. trypodendri were collected from hardwood-infesting bark beetles in Poland (Jankowiak et al. 2017, while three other Leptographium species have been isolated and formally described from hardwood wounds (Jankowiak et al. , 2019c. There was no information on D. alni-associated fungi before 2019. Recent Polish research reported that only L. alneum (named as Leptographium sp. 7) is an associate of D. alni (Jankowiak et al. 2019a). However, the additional isolations conducted in 2018 demonstrated that this beetle species apart from L. alneum, was also associated with other ophiostomatoid species including C. synnemata and L. piriforme. Among them, only C. synnemata and L. alneum were commonly found in association with D. alni. The common occurrence of these species suggests their important role as fungal associates of D. alni in Poland. The results of the present study and other Polish findings (Jankowiak et al. 2019a) indicated that C. synnemata and L. alneum have been found only occasionally from other beetle species and therefore can be considered as regular and possible specific associates of D. alni. Leptographium piriforme is less specific and can be found with other beetle species (Jankowiak and Kolařík 2010) or hardwood wounds (Jankowiak et al. 2019c) in low numbers.
This work represents the most detailed survey of Ophiostomatales associated with D. alni in Europe. Two new species were described. Ophiostomatoid fungi on hardwoods have been relatively well investigated in Poland (Jankowiak et al. 2017(Jankowiak et al. , 2019aAas et al. 2018), but in other parts of Europe they are still poorly studied. In addition, many ophiostomatoid species have not been formally described and our study has contributed to filling this knowledge gap. The findings of this study clearly showed that the diversity and taxonomic placement of many members of the Ophiostomatales associated with hardwoods-infesting bark beetles in Europe are still poorly understood.