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 twenty-eight 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-five 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).
Phylogenetic Analyses
Alignments for the ITS1-5.8S-ITS2 data set contained 738 characters; for the ITS2-28S data 496 characters; for the CAL data 613 characters; for the TEF1-α data 785 characters; for the βT data 526 characters for the ITS1-5.8S-ITS2-28S+ βT data 1839 characters (including gaps). The exon/intron arrangement of the CAL data included intron 3, exon 4, intron 4, exon 5 intron 5, exon 6 and intron 6. The exon/intron arrangement of the TEF1-α data included intron 3, exon 4, intron 4 and exon 5. The aligned βT gene region consisted of exons 3 to 6, interrupted by introns 3, 4 and 5.
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 (Figs. 2-6). 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 defined 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 (Figs. 2-6). 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).
The analyses of the combined ITS1-5.8S-ITS2-28S+βT datasets clearly separated Taxa 1 to 4 from the other known species of Ceratocystiopsis and also from each other (Fig. 7). Nineteen isolates of taxon 5 grouped with the ex-type isolate of C. rollhanseniana (UM113) and other isolates of this species based on ITS2-28S (Fig. 3). The βT , CAL and TEF1-α data also confirmed that those isolates represented C. rollhanseniana (Figs. 4-6). The ITS1-5.8S-ITS2, ITS2-28S, βT, CAL and TEF1-α datasets confirmed that four isolates of taxon 6 grouped with ex-type isolate of C. neglecta (Figs. 2-6).
Taxonomy
The morphological characterization and phylogenetic comparisons based on five genetic loci, showed that four herein described morphotypes (taxon 1, 2, 3 and 4) are distinct from each other and from other known taxa in the genus Ceratocystiopsis. Therefore, they are described here as new species:
Ceratocystiopsis chalcographii Jankowiak & H. Solheim, sp. nov.
MycoBank: 841844
(Fig. 8)
Etymology: The epithet chalcographii (Latin), refers to the species name of the bark beetle vector of this fungus, Pityogenes chalcographus.
Diagnosis: This species is closely related to C. minuta and C. norroenii. Ceratocystiopsis chalcographii has longer perithecial necks (36–124 μm) compared to C. minuta (20–45 μm) (Reid and Hausner 2010). Their ostiolar hyphae is also longer (7.7–16.4 vs. to 12 µm) just as the length of the ascospores (9.9–15 vs. 10–13 µm) (Reid and Hausner 2010). Ceratocystiopsis chalcographii has obovoid conidia, whereas C. minuta has oblong conidia (Reid and Hausner 2010). Ceratocystiopsis chalcographii has longer conidia (2.9–6 μm) compared to C. minuta (2–4 μm) (Reid and Hausner 2010). In addition, conidiophores of C. chalcographii are simple or slightly branched while C. minuta produces simple or verticillate to irregular branched conidiophores (Reid and Hausner 2010). Ceratocystiopsis norroenii also produces branched conidiophores, however this taxon could be distinguished from C. minuta by oblong and larger conidia (2.7–5 × 1.1–2.3 vs. 2 –4 ×1.2 µm). In addition, C. norroenii does not produce a sexual morph.
Type: Norway, Nannestad, from Pityogenes chalcographus infesting Picea abies, 15 Oct. 2014, H. Solheim and M.E. Waalberg (O-F-259424 – holotype; CBS 147954 = CMW 57280 – ex-type culture).
Description: Sexual morph perithecial. Perithecia abundantly produced on media and sterilized spruce twigs, various in shape, bases black, globose or sub-prolate (46–)55–101(–125) µm diam. Necks black, straight or slightly curved, (36–)55–88(–124) µm long. Diameter of the necks (5.9–)8.2–13(–17) µm at the apex and (20.3–)23.9–32.3(39.7) µm at the base. Ostiolar hyphae present, hyaline, aseptate, straight, tapering extensively towards the apex, 5–8 in number, (7.7–)9.2–13(–16.4) µm long. Asci not observed. Ascospores one-celled, hyaline, including the sheath falcate in side view (9.9–)11.6–14(–15) × (1–)1.2–1.6(–1.8) µm, accumulated in a white-coloured mass at the tip of the neck.
Asexual morphs: hyalorhinocladiella-like.
Hyalorhinocladiella-like morph: conidiophores mononematous, micronematous or semi-macronematous, arising from vegetative hyphae, simple or slightly branched, upright, straight or undulate, producing conidiogenous cells at their apices, (1.7–)6.9–23.4(–40.4) × (0.7–)1–1.8(–2.2) µm. Conidiogenous cells integrated, hyaline, blastic, not denticulate. Conidia hyaline, aseptate, obovoid with the upper part swollen, apex round, tapering toward base, sometimes pyriform, no sheath, (2.9–)3.6–5(–6) × (0.9–)0.9–1.2(–1.9) µm, no sheath.
Cultures: Colonies with optimal growth at 30°C on 2% MEA with radial growth rate 1.11 (± 0.05) mm/d. Colonies white, margin smooth, floculose, with white lumps of slimy conidia. Hyphae hyaline to pale grey in colour, reverse pale orange (Kornerup and Wanscher 1978), smooth, submerged in the medium and aerial mycelium abundant, not constricted at the septa, 1–2.6 (mean 1.7±0.4) µm diam., asexual morph very abundant.
Ecology: Isolated from Pityogenes chalcographus infesting Picea abies and Pinus sylvestris.
Habitat: Picea abies and Pinus sylvestris forests.
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 (Figs. 2-4). They can be both differentiated from C. minuta by the morphology of sexual morph as well as morphology of the conidia and conidiophores.
Ceratocystiopsis chalcographii was rarely isolated from bark beetles infesting P. abies and P. sylvestris (Table 3). It was found only in association with P. chalcographus on Scots pine in Rendalen and on Norway spruce in Nannestad. This species is one of two Ceratocystiopsis species isolated from P. chalcographus (Taxa 1 and 3).
Additional specimen examined: Norway: Rendalen, from Pityogenes chalcographus infesting Pinus sylvestris, 29 Oct. 2014, H. Solheim, (O-F-259425, culture CBS 147955 = CMW 57282).
Ceratocystiopsis norroenii Jankowiak & H. Solheim, sp. nov.
MycoBank: 841845
(Fig. 9)
Etymology: The epithet norroenii (Latin), refers to the word Norrøn, which in norse mythology means from north, 49 out of 50 specimens collected during this study are from the northernmost part of Norway, Troms and Finnmark.
Diagnosis: See comparisons between C. norroenii and C. minuta under C. chalcographii.
Type: Norway, Karasjok, from Ips acuminatus infesting Pinus sylvestris, 21 Jun. 2015, H. Solheim (O-F-259426 – holotype; CBS 147956 = CMW 57283 – ex-type culture).
Description: Sexual morph: not observed.
Asexual morphs: hyalorhinocladiella-like.
Hyalorhinocladiella-like morph: conidiophores micronematous or macronematous. The micronematous conidiophores, arising from vegetative hyphae, simple or branched (1 branches), hyaline, upright, straight or undulate, producing conidiogenous cells at their apices (1.4–)12.1–28.1(–33.3) × (0.9–)1.2–1.7(–2.1) µm, often conidia formed singly, either directly on the side of vegetative hyphae or on short lateral branches. The macronematous conidiophores, hyaline, (9.3–)29.6–48.2(–64.9) long, and (8.5–)11.2–19.4(–15.8) µm wide at the apex, arising from a basal cell, (4.3–)6.2–11.7(–16) × (1.4–)1.7–2.6(–3.1) µm. The basal cells branch irregularly or penicillate and form 1-2 branches producing conidiogenous cells at their apices, 3–4 per branch. Conidiogenous cells integrated, hyaline, blastic, sometimes denticulate. Conidia hyaline, smooth, unicellular, oblong, sometimes slightly obovoid (2.7–)3.2–4.1(–5.1) × (1.1–)1.2–1.6(–2.3) µm, no sheath.
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.
Ecology: Isolated from Ips acuminatus and Pityogenes quadridens infesting Pinus sylvestris.
Habitat: Pinus sylvestris forests.
Distribution: Currently only known from Norway.
Notes: See comparisons between C. norroenii and C. minuta under C. chalcographii. Ceratocystiopsis norroenii was commonly isolated from Ips acuminatus and Pityogenes quadridens infesting Scots pine growing in the north part of Norway.
Additional specimen examined: Norway: Målselv, from Ips acuminatus infesting Pinus sylvestris, 21 Jun. 2015, H. Solheim (O-F-259427, culture CBS 147957 = CMW 57284).
Ceratocystiopsis debeeria Jankowiak & H. Solheim, sp. nov.
MycoBank: 841846
(Fig. 10).
Etymology: Named for Prof. Wilhelm de Beer who has contributed greatly to the fungal taxonomy of ophiostomatoid species.
Diagnosis: Ceratocystiopsis debeeria is the closest phylogenetic relative to C. neglecta. It can be differentiated from C. neglecta by the absence of perithecia and producing simple or very slightly irregularly branched conidiophores. Ceratocystiopsis neglecta produces simple or strongly penicilliaty branched conidiophores (Kirschner and Oberwinkler 1999). Morphologically, these two species also differ by the size of their conidia ranging from 3–4.5 x 1–1.5 µm in C. neglecta (Kirschner and Oberwinkler 1999) vs. 2.7–5.2 × 0.9–1.7 µm in C. debeeria.
Type: Norway, Flesberg, from Pityogenes chalcographus infesting Picea abies, 31. Oct. 2014, M.E. Waalberg and R.A. Lindseth (O-F-259428 – holotype; CBS 147958 = CMW 57288 – ex-type culture).
Description: Sexual morph: not observed.
Asexual morphs: hyalorhinocladiella-like.
Hyalorhinocladiella-like morph: conidiophores mononematous, micronematous, arising from vegetative hyphae, simple or slightly and irregularly branched, upright, straight or undulate, producing conidiogenous cells at their apices, (7.8–)10.6–25.8(–37.9) × (0.8–)1–1.4(–1.8) µm. Conidiogenous cells integrated, hyaline, blastic, rarely denticulate. Conidia hyaline, aseptate, obovoid with the upper part swollen, apex round, tapering toward base, sometimes oblong, no sheath, (2.7–)3.1–4.2(–5.2) × (0.9–)1.1–1.4(–1.7) µm.
Cultures: Colonies with optimal growth at 25°C on 2% MEA with radial growth rate 0.82 (± 0.18) mm/d. Colonies yellowish white, margin smooth, with suede-like surface. Hyphae hyaline to pale grey in colour, reverse pale orange (Kornerup and Wanscher 1978), smooth, submerged in the medium and aerial mycelium rarely, not constricted at the septa, 0.8–2.9 (mean 1.6±0.4) µm diam., asexual morph very abundant.
Ecology: Isolated from Pityogenes chalcographus infesting Picea abies and Pinus sylvestris.
Habitat: Picea abies and Pinus sylvestris forests.
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 Flesberg.
Additional specimen examined: Norway, Rendalen, from Pityogenes chalcographus infesting Pinus sylvestris, 29 Oct. 2014, H. Solheim (O-F-259429, culture CBS 147959 = CMW57290).
Ceratocystiopsis troendelagii Jankowiak & H. Solheim, sp. nov.
MycoBank: 841847
(Fig. 11)
Etymology: The epithet trendelagii (Latin) refers to the area in mid-Norway, Trøndelag, from which this fungus was collected.
Type: Norway, Lierne, from Polygraphus subopacus infesting Picea abies, 8 Aug. 2015, T. Kvamme and Å. Lindelöv (O-F-259430 – holotype; CBS 147962 = CMW57294 ex-type culture).
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.
Description: Sexual morph perithecial. Perithecia abundantly produced on media and sterilized spruce twigs, bases black, globose or sub-globose (25–)33–45(–54) µm diam. Necks black, straight or slightly curved, variable in length, (9–)11–29(–72) µm long. Diameter of the necks (4–)4.7–7.7(–10.7) µm at the apex and (12.5–)14.9–20.1(24.6) µm at the base. Ostiolar hyphae present, hyaline, aseptate, straight, tapering extensively towards the apex, 3–6 in number, (3.9–)7.2–10.5(–11.8) µm long. Asci not observed. Ascospores one-celled, hyaline, including the sheath falcate in side view (7–)8.8–11.8(–13.5) × (0.7–)0.8–1.1(–1.3) µm, accumulated in a white-coloured mass at the tip of the neck.
Asexual morphs: hyalorhinocladiella-like.
Hyalorhinocladiella-like morph: conidiophores micronematous or macronematous. The micronematous conidiophores, arising from vegetative hyphae, simple, hyaline, upright, straight or undulate, producing conidiogenous cells at their apices (5.9–)8.7–20.4(–30.4) × (0.8–)1.3–2.2(–2.7) µm. The macronematous conidiophores, hyaline, (17.8–)21.9–34.8(–41.2) long, and (4.4–)11.7–28.8(–37.7) µm wide at the apex, arising from a basal cell, (3.5–)5.1–10.9(–16.5) × (1.7–)2.2–3.3(–4.1) µm. The basal cells branch irregularly and form 2-3 branches producing conidiogenous cells at their apices, 2–4 per branch. Conidiogenous cells integrated, hyaline, blastic, sometimes denticulate. Conidia hyaline, smooth, unicellular, oblong or obovate (2–)2.6–3.5(–4.1) × (0.8–)1.1–1.8(–2.2) µm, no sheath.
Cultures: Colonies with optimal growth at 20°C on 2% MEA with radial growth rate 0.72 (± 0.05) mm/d. Colonies yellowish 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, 0.7–2.2 (mean 1.2±0.4) µm diam., asexual morph very abundant.
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.
Additional specimen examined: Norway, Lierne, from Pityogenes saalasi infesting Picea abies, 8 Aug. 2015, T. Kvamme and Å. Lindelöv (O-F-259431, culture: CBS 147963 = CMW57295).