Distribution of the Genus Ceratocystiopsis (Ascomycota, Ophiostomatales) on Conifers in Norway, With Description of Four New Species

The genus Ceartocystiopsis includes ascomycetes fungi belonging to the Ophiostomatales that are not well recognized in the world. Described species so far have been found mainly in association with bark beetles in the Northern Hemisphere. The aims of this study were therefore to survey of distribution of species of Ceratocystiopsis associated with bark beetles infesting Picea abies and Pinus sylvestris in Norway. Adults for 22 different bark beetle species were collected from 13 stands in Norway. During this study, we recovered 126 isolates showing anities to Ceratocystiopsis representing six species, including two described and four undescribed taxa. The four undescribed taxa collected during this work were characterised based on their morphological characteristics and multi-gene phylogenies. Herein, we describe these new species as Ceratocystiopsis chalcographii sp. nov., Ceratocystiopsis debeeria sp. nov., Ceratocystiopsis norroenii sp. nov. and Ceratocystiopsis troendelagii sp. nov. Ceratocystiopsis norroenii and C. rollhanseniana were the most frequently isolated species although the latter species had a much wider vector range. This study expands our knowledge about the taxonomy and species diversity of Ceratocystiopsis and beetle-fungus relationships. two isolates Agar disks 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 of the six putative new species were incubated at temperatures between 5, and 35 °C at 5 °C intervals. The radial growth (two measurements perpendicular to each other per plate) was determined 14 d after inoculation, and growth rates were calculated as mm/d. temperatures on 2% MEA. Ceratocystiopsis norroenii and C. debeeria grew optimally at 25 °C, C. chalcographii at 30 °C, and C. troendelagii at 20 °C. The four species described in this study can easily be distinguished from each other and from the other species of Ceratocystiopsis based on DNA sequence comparisons. Analyses of the ITS region sequence data were already sucient to distinguish these species. Most of the formally described species of Ceratocystiopsis are known only from conifers although C. lunata and C. synnemata were recently collected from hardwood-infesting beetles (Li et al. 2018; Strzałka et al. 2020; Nel 2021). The results of the present study conrm that in general there is a close anity of Ceratocystiopsis species to conifers.


Introduction
Hylastes brunneus, Hylastes cunicularis, Hylurgops palliatus, Ips acuminatus, Ips sexdentatus, Orthotomicus laricis, Orthotomicus proximus, Orthotomucus suturalis, Pityogenes bidentatus, Pityogenes chalcographus, Pityogenes quadridens, Pityogenes saalasi, Pityophthorus micrographus, Polygraphus poligraphus, Polygraphus subopacus, Tomicus piniperda and Tryopdendron lineatum. The bark beetles were collected by hand from Picea abies and Pinus sylvestris. Bark beetle adults were excised from the bark and galleries with sterilized tweezers and stored individually in sterile 1.5 ml Eppendorf tubes for later fungal isolations. Each bark beetle was divided into three parts, elytra, head and the rest, before placing the parts in three separate Petri dishes with malt extract agar [MEA; 6.25 g malt Bacto™ agar powder (Beckton, Dickinson, Sparks, USA), 10 g agar (bactoagar powder from VWR International, Leuven, Belgium), 0.5 l deionized water] without any cycloheximide. Emerging cultures were puri ed by transferring small pieces of mycelium or spore masses from individual colonies to fresh MEA. Fungi were incubated at 20 °C, then subcultured onto 2% MEA and stored at 4 °C. After two weeks of incubation, the puri ed fungal cultures were grouped into morphotypes based on their morphological characteristics. Depending on the number of isolates belonging to the same morphotype, 2-19 isolates per morphotype were chosen for molecular identi cation ( Table 2).
The cultures are maintained in the culture collection of Norwegian Institute of Bioeconomy (NIBIO), Norway. Ex-type isolates of new species described in this study were deposited in the collection of 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, Pretoria, South Africa. Type specimens were deposited in the Mycological Herbarium (O), Natural History Museum, University of Oslo, Norway. The types of C. neglecta and C. rollhanseniana were sourced from the culture collection of the Westerdijk Fungal Biodiversity Institute (CBS), and from the culture collection of the University of Manitoba (WIN) in Canada (Table 2). Taxonomic descriptions and nomenclatural data were registered in MycoBank (www.MycoBank.org) (Robert et al. 2013).

Microscopy and growth studies
Morphological characters were examined for selected isolates as well as for the herbarium specimens selected as types. Isolates were grown on 2% Malt Extract Agar (MEA) (20 g Bacto™ malt extract, 20 g agar Bacto™ agar powder (Becton Dickinson and Company, Franklin Lakes, USA) in 1 l deionized water). In attempts to induce the formation of ascomata, autoclaved twigs of host trees including the bark were placed at the centres of agar plates containing MEA.
Fungal cultures were derived from single conidia. To promote the production of ascomata, single conidial isolates were crossed in all possible combinations, following the technique described by Grobbelaar et al. (2009). These cultures were incubated at 25 o C and monitored regularly for the appearance of fruiting structures.
Morphological features were examined by mounting fungal tissue in 80% lactic acid on glass slides, and fruiting 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.
Microscopy followed the technique described by Kamgan Nkuekam et al. (2011). Colour designations were based on the colour charts of Kornerup and Wanscher (1978).
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 novel species were determined by analysing the radial growth for two isolates (two for each species). Agar disks (5 mm diam.) 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 of the six putative new species were incubated at temperatures between 5, and 35 °C at 5 °C intervals. The radial growth (two measurements perpendicular to each other per plate) was determined 14 d after inoculation, and growth rates were calculated as mm/d.
MP analyses were performed using PAUP* 4.0b10 (Swofford 2003). Gaps were treated as fth state. Bootstrap analysis (1000 bootstrap replicates) was conducted to determine the levels of con dence 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). Four MCMC chains were run in parallel for 10 million generations applying the best-t model for each dataset. Trees were sampled every 100 th generations, resulting in 100,000 trees. Tracer v1.4.1 (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.

Collections of bark beetles and fungi
Altogether 22 bark beetle species were collected from Norway during this study (Table 3). Among the bark beetles carrying Ceratocystiopsis species only P. chalcographus occurred on both pine and spruce. Five bark beetle species with Ceratocystiopsis species were found on pine, while four bark beetle species were encountered only on spruce. The isolations from bark beetles yielded a total of 126 isolates resembling Ceratocystiopsis spp.
In total, 126 Ceratocystiopsis spp. isolates were obtained from 459 sampled beetles (Table 3). Fifty isolates were collected from I. acuminantus, 21 isolates were collected from O. proximus, 15 isolates were collected from P. chalcographus, and 12 isolates were collected from C. cinereus. The additional twentyeight isolates were collected from H. palliatus, P. saalasi, Pol. subopacus, C. pusillus, P. bidentatus, and P. quadridens (Table 3). Based on morphological observations the fungal isolates obtained from this study could be arranged into six taxa. Fifty-ve isolates represented taxon 6, were isolated from six different bark beetles. This taxon was the most frequently encountered on P. bidentatus (72%) and O. proximus (70%). The frequency of occurrence of this taxon on C. cineraeus was also relatively high (40%). Fifty isolates belonged to taxon 2. This taxon was found only in high frequency on I. acuminatus (54%). The remaining 16 isolates represented four Ceratocystiopsis taxa and were collected from 1 or 2 different beetle species (Table 3). The frequency of occurrence of these species was less than 11% (Table 3).
DNA sequence data were obtained for 45 isolates representing all six morphological groups represented among the 126 Ceratocystiopsis resembling fungal isolates recovered from 459 sample beetle specimens (Table 2). BLAST analyses of the ribosomal DNA sequences placed the examined isolates in the genus Ceratocystiopsis. Sequences from our 45 representative strains when combined with those of other members of the genus Ceratocystiopsis formed six independent well-supported terminal clades representing six phylogenetic species (Taxa 1-6) in the ITS1-5.8S-ITS2, ITS2-28S, βT, CAL and TEF1-α phylogenetic inferences . Based on the phylogenetic analyses of the ITS1-5.8S-ITS2 and ITS2-28S data sets (Figs. 2-3), the isolates emerged as two known and four undescribed taxa of the genus Ceratocystiopsis.
Four isolates recovered from P. abies and P. sylvestris infested by P. chalcographus (i.e., taxon 1) in the ITS1-5.8S-ITS2 and ITS2-28S trees grouped in the "C. minuta" clade C as de ned by Plattner et al. (2009), this clade includes European and Japanese strains of Ceratocystiopsis minuta s.l. In the βT tree, eight isolates of taxon 1 recovered in this study clustered into a distinct monophyletic clade next to a Ceratocystiopsis strain (CMW4352) from Austria and Japanese isolates of Ceratocystiopsis spp. (YCC330 and YCC329) (Fig. 4). Although only poorly supported this clade appears to share a common node with strains assigned to C. minuta s. str. (including UM1532) and C. weihaiensis (Fig. 4).
Nine isolates obtained from P. sylvestris infested by I. acuminatus (i.e., taxon 2) in the ITS1-5.8S-ITS2, ITS2-28S, βt, CAL, and TEF1-α trees (Figs. 2-6) form a well-supported lineage, distinct from all the other known species of Ceratocystiopsis. Six isolates representing taxon 3 and taxon 4 also form a well-supported lineages distinct from other known species of Ceratocystiopsis . Taxon 3 appears to represent a phylogenetically distinct lineage, potentially sharing ancestry with C. neglecta (Figs. 2-4), however the nodes supporting monophyly are only poorly supported. Members of taxon 4 form a linage adjacent to C. minima within the ITS2-28S sequences based phylogenetic tree (Fig. 3), however the other dataset do not support monophyly for these two species (Figs. 2, 4 and 7).
Distribution: Currently only known from Norway.
Notes: Ceratocystiopsis chalcographii and C. norroenii formed two distinct, well-supported clades within the C. minuta group, in which they were closely related to C. minuta . They can be both differentiated from C. minuta by the morphology of sexual morph as well as morphology of the conidia and conidiophores.
Cultures: Colonies with optimal growth at 25°C on 2% MEA with radial growth rate 1.11(± 0.15) mm/d. Colonies orange white, margin smooth, with suede-like surface, but occasionally funiculose. Hyphae hyaline, reverse pale orange (Kornerup and Wanscher 1978), smooth, submerged in the medium and aerial mycelium rarely, not constricted at the septa, 0.5-1.8 (mean 1±0.3) µm diam., asexual morph very abundant. Etymology: Named for Prof. Wilhelm de Beer who has contributed greatly to the fungal taxonomy of ophiostomatoid species.
Distribution: Currently only known from Norway.
Notes: In a phylogenetic perspective, C. debeeria is closely related to C. neglecta (Figs. 2-4). Ceratocystiopsis debeeria is characterized by the absence of perithecia, which are present in C. neglecta (Kirschner and Oberwinkler 1999). In addition, C. debeeria can be distinguished from C. neglecta by the size of conidia.
Ceratocystiopsis debeeria was only isolated on a few occasions from Pityogenes chalcographus infesting Picea abies in Rendalen and Pinus sylvestris in Diagnosis: The species is characterized by perithecia and a hyalorhinocladiella-like morph. It can be differentiated from the closely related species C. minima by smaller perithecial base diameter 25-54 µm vs. 40-85 µm (Olchowecki and Reid 1974). Also C. troendelagii has shorter conidia (2.7-5.2 µm) compared to C. minima (2.5-8.0) µm (Olchowecki and Reid 1974). In addition, the conidia of C. minima are clavate or oblong while C. troendelagii has mainly obovoid conidia.

Ecology: Isolated from Pityogenes saalasi and Polygraphus subopacus infesting Picea abies and Pinus sylvestris
Habitat: Picea abies and Pinus sylvestris forests Distribution: Currently only known from Norway.
Notes: Ceratocystiopsis troendelagii is phylogenetically close to C. minima but formed a distinct clade in both the ITS1-5.8S-ITS2 and ITS2-28S trees as well in the βT based tree (Figs. 2-4). It can be differentiated from C. minima by dimension of the perithecial base as well as morphology of conidia.
Ceratocystiopsis troendelagii was the rarest Ceratocystiopsis species isolated in this study. It was isolated twice in the same plot, once from Pityogenes saalasi and once from Polygraphus subopacus. Both bark beetles seem to have a northern distribution in Fennoscandia.

Discussion
In the present study, we collected 22 species of bark beetles from Picea abies and Pinus sylvestris located in Norway. From these beetles, we recovered 126 isolates that based on morphology showed a nities towards the genus Ceratocystiopsis. Analyses of molecular and morphological data indicated that four out of the six Ceratocystiopsis species recovered in this study were previously undescribed. Herein, we described these new species as C. chalcographii, C. debeeria, C. norroenii and C. troendelagii. Description of these new species brings the total number of recognized species in this genus to 24, of which seven occur in Norway. These include the six species recovered in this study as well as C. minuta, which was previously found in association with I. typographus (Solheim 1986(Solheim , 1992(Solheim , 1993. In addition, C. rollhanseniana was described by Hausner et al. (2003) based on the Norwegian isolates collected by James Reid from P. sylvestris.
Ceratocystiopsis norroenii and C. rollhanseniana were the most frequently isolated Ceratocystiopsis species in this study. However, these species appear to have different distribution and insect associations. Ceratocystiopsis norroenii appears to be a consistent associate of Ips acuminatus on P. sylvestris while C. rollhanseniana has broader host/insect associations including different bark beetle species found on P. abies and P. sylvestris. Our phylogenetic analysis of the ITS2-28S sequences (Fig. 3) showed that some Polish isolates previously identi ed as C. minuta and collected from P. abies infested by I. typographus (CBS 116963, CBS 116796) actually represented C. rollhanseniana, showing its wider distribution in Europe.
Ceratocystiopsis norroenii belongs to the C. minuta s.l. grouping that includes several lineages from Europe and Japan (Plattner et al. 2009). According to Plattner et al. (2009) C. minuta contains different phylogenetic species, which are well-de ned based on molecular and to some degree morphological criteria. Therefore, to stabilize the taxonomic position of C. minuta an epitype was designated for C. minuta based on the Polish strain RJ705=UAMH 11218 = WIN(M) 1532 by Reid and Hausner (2010). The genetic linage of C. norroenii was not represented in Plattner et al.'s study (2009) suggesting that here are many C. minuta-like species that are morphologically similar. For example, C. weihaiensis a species from China was shown to be phylogenetically close to C. minuta (Chang et al. 2021).
In this study, besides C. norroenii we recovered eight other isolates that are part of the C. minuta s.l. species complex. These isolates were recovered from P. chalcographus on P. abies and represented a new species designated as C. chalcographii. The ITS1-5.8S-ITS2, ITS2-28S and βT sequences of this taxon were identical or very close to several isolates from Scotland, Austria and Japan, which were identi ed previously as C. minuta. Therefore, C. chalcographii probably has a wider geographical distribution in Europe and Asia. More studies are needed on the taxonomic/genetic diversity of the C. minuta s.l. complex to resolve the various cryptic species that might be part of this group. In this study, P. chalcographus was also associated with C. debeeria. However, this fungus was found only in association with this beetle species, suggesting that C. debeeria is a speci c ectosymbiont of P. chalcographii.
Ceratocystiopsis troendelagii and Ceratocystiopsis neglecta were found only on a few occasions in association with conifer-infesting bark beetles in Norway. Ceratocystiopsis troendelagii seems to be a fungal associate of P. saalasi and P. subopacus while C. neglecta was found associated with H. palliatus and P. subopacus. Ceratocystiopsis neglecta so far was only known from studies on different bark-and ambrosia beetles infesting coniferous trees in Germany (Kirschner 2001;Kirschner and Oberwinkler 1999). This is the rst report of C. neglecta as a fungal associate of P. subopacus.
All of the species described in this study are morphologically similar, having a hyalorhinocladiella-like asexual states with hyaline conidia produced on nondenticulate conidiogenous cells. Ceratocystiopsis norroenii and C. troendelagii formed only simple conidiophores while C. chalcographii and C. troendelagii formed simple as well as condiophores with irregular branching patterns resembling loose structures. Where ascomata were present, these tended to have globose or elongated bases with short necks terminating in ostiolar hyphae and falcate acospores surrounded by hyaline sheaths. The newly described species demonstrated different optimal growth temperatures on 2% MEA. Ceratocystiopsis norroenii and C. debeeria grew optimally at 25 °C, C. chalcographii at 30 °C, and C. troendelagii at 20 °C. The four species described in this study can easily be distinguished from each other and from the other species of Ceratocystiopsis based on DNA sequence comparisons. Analyses of the ITS region sequence data were already su cient to distinguish these species.
Most of the formally described species of Ceratocystiopsis are known only from conifers although C. lunata and C. synnemata were recently collected from hardwood-infesting beetles (Li et al. 2018;Strzałka et al. 2020;Nel 2021). The results of the present study con rm that in general there is a close a nity of Ceratocystiopsis species to conifers.
In this study, six species of Ceratocystiopsis were isolated from 22 species of bark beetles. Based on morphological characters and DNA sequence data, four new species of Ceratocystiopsis were described in addition this is the rst report of C. neglecta for Norway. Although the relationships between fungi and bark beetles are relatively well recognized in Europe our survey led to the discovery and description of new species, suggesting that there are more novel fungal taxa to be discovered. The results of this study have substantially expanded our knowledge of Ceratocystiopsis species living in northern parts of Europe. Broadly, the results suggest that Ceratocystiopsis species are frequently occurring members of the Ophiostomatales in conifer ecosystems in Norway.  Tables   Table 1 Bark beetle sampling sites Picea abies (Pa), Pinus sylvestris (Ps)   Figure 1 The sample sites in Norway where isolations from bark beetles yielded Ceratocystiopsis species.