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Research Article
Taxonomy and phylogeny of the Leptographium olivaceum complex (Ophiostomatales, Ascomycota), including descriptions of six new species from China and Europe
expand article infoMingliang Yin‡§, Michael J. Wingfield§, Xudong Zhou|, Riikka Linnakoski§, Z. Wilhelm de Beer#
‡ South China Agricultural University, Guangzhou, China
§ University of Pretoria, Pretoria, South Africa
| University of Pretoria, Pretoria, China
¶ Natural Resources Institute Finland, Helsinki, Finland
# University of Pretoria, Preotoria, South Africa

Corresponding author: Mingliang Yin ( mingliang.yin@scau.edu.cn )

Academic editor: Danny Haelewaters
© 2019 Mingliang Yin, Michael J. Wingfield, Xudong Zhou, Riikka Linnakoski, Z. Wilhelm de Beer.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Yin M, Wingfield MJ, Zhou X, Linnakoski R, de Beer ZW (2019) Taxonomy and phylogeny of the Leptographium olivaceum complex (Ophiostomatales, Ascomycota), including descriptions of six new species from China and Europe. MycoKeys 60: 93-123. https://doi.org/10.3897/mycokeys.60.39069
Open Access

Abstract

The Leptographium olivacea complex encompasses species in the broadly defined genus Leptographium (Ophiostomatales, Ascomycota) that are generally characterized by synnematous conidiophores. Most species of the complex are associates of conifer-infesting bark beetles in Europe and North America. The aims of this study were to reconsider the delineation of known species, and to confirm the identity of several additional isolates resembling L. olivacea that have emerged from recent surveys in China, Finland, Poland, Russia, and Spain. Phylogenetic analyses of sequence data for five loci (ACT, TUB, CAL, ITS2-LSU, and TEF-1α) distinguished 14 species within the complex. These included eight known species (L. cucullatum, L. davidsonii, L. erubescens, L. francke-grosmanniae, L. olivaceum, L. olivaceapini, L. sagmatosporum, and L. vescum) and six new species (herein described as L. breviuscapum, L. conplurium, L. pseudoalbum, L. rhizoidum, L. sylvestris, and L. xiningense). New combinations are provided for L. cucullatum, L. davidsonii, L. erubescens, L. olivaceum, L. olivaceapini, L. sagmatosporum and L. vescum. New Typifications: Lectotypes are designated for L. olivaceum, L. erubescens and L. sagmatosporum. Epitypes were designated for L. olivaceapini and L. sagmatosporum. In addition to phylogenetic separation, the synnematous asexual states and ascomata with almost cylindrical necks and prominent ostiolar hyphae, distinguish the L. olivaceum complex from others in Leptographium.

Keywords

bark beetle, Leptographium, integrative taxonomy, new species, Ophiostomatales, phylogeny

Introduction

Species of Leptographium are commonly associated with bark beetles and weevils, and are responsible for causing sapstain on a wide range of primarily coniferous trees (Jacobs and Wingfield 2001). The genus also includes some important tree pathogens such as species in the Leptographium wageneri complex that cause black stain root disease (Goheen and Hansen 1978). In their monograph of Leptographium, Jacobs and Wingfield (2001) treated the asexual states of 46 species in the genus, all characterized by mononematous conidiophores branched at their apices. Conidia aggregate in slimy droplets at the apices of these structures, which make these species well-adapted for arthropod dispersal.

Following the “one fungus one name” principles adopted in the Melbourne Code (Hawksworth 2011), De Beer and Wingfield (2013) re-evaluated the taxonomy of Leptographium, considering available DNA sequence data for all species. Ninety-four species were included and ten species complexes were defined within a broadly defined concept for Leptographium sensu lato, based on phylogenies resulting from ribosomal internal transcribed spacer (ITS) and partial LSU sequences.

One of the species complexes recognized in Leptographium s.l. by De Beer and Wingfield (2013) was the L. olivaceum complex. Earlier, Zipfel et al. (2006) had shown that L. olivaceum produces synnematous asexual states, which is unlike mononematous conidiophores traditionally defining Leptographium. In extended phylogenies, Massoumi Alamouti et al. (2007), Six et al. (2011), and Linnakoski et al. (2012) showed that additional species with synnematous asexual states grouped in a monophyletic lineage with L. olivaceum. Six et al. (2011) referred to this lineage as the L. olivaceum species complex for the first time and they included L. olivaceum (Mathiesen-Käärik, 1951), L. sagmatosporum (Wright & Cain, 1961), L. olivaceapini (Davidson, 1971), and L. cucullatum (Solheim, 1986) in their phylogeny. Subsequently, L. davidsonii (Olchowecki & Reid, 1974) and L. vescum (Davidson, 1958) were shown to also belong to this complex (Linnakoski et al. 2012, De Beer and Wingfield 2013).

The six species currently residing in the L. olivaceum complex have morphologically similar sexual and asexual states. They produce globose ascomata with long, nearly cylindrical necks, terminating in prominent ostiolar hyphae on which sticky droplets are formed that contain orange-section shaped ascospores with cucullate gelatinous sheaths (Mathiesen-Käärik 1951, Davidson 1958, Wright and Cain1961, Davidson 1971, Olchowecki and Reid 1974, Solheim 1986). This study includes isolates representing all species in the L. olivaceum complex as well as morphologically similar isolates from recent surveys of fungi in China, Europe, and Russia. The aims of the study were to reconsider and redefine the species boundaries in the L. olivaceum complex based on phylogenetic analyses of multilocus regions, to provide neotypes for species where type specimens have been lost or are inadequate, and to describe new species in this complex.

Methods

Isolates

All isolates included in this study are listed in Table 1. Reference isolates were obtained from the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa. Ex-type isolates of newly described species were deposited in the Westerdijk Fungal Biodiversity Institute (CBS), Utrecht, in the Netherlands. Type specimens of new species were deposited in the National Collection of Fungi (PREM), Pretoria, South Africa. Taxonomic novelties and new typification events for known taxa were registered in MycoBank (Robert et al. 2013).

Table 1.
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Isolates used in the present study.

Species1 Isolate no.2 Country Host Insect GenBank accession no. 3
CMW no. CBS no. ITS2-LSU ACT TUB CAL TEF-1α
Leptographium breviscapum 38888 H 136507 China Picea crassifolia Polygraphus poligraphus MN516697 MN517641 MN517672 MN517707 MN517742
38889 P 136508 China Picea crassifolia Polygraphus poligraphus MN516698 MN517642 MN517673 MN517708 MN517743
38890 China Picea crassifolia Polygraphus poligraphus MN516699 MN517643 MN517674 MN517709 MN517744
L. conplurium 23289 P 128834 Finland Picea abies Dryocoetus autographus MN516701 MN517644 JF279994 MN517710 MN517745
23295 Finland Picea abies Dryocoetus autographus MN516702 MN517645 JF279993 MN517711 JF280036
23315 H 128923 Finland Picea abies Dryocoetus autographus MN516700 MN517646 JF279989 MN517712 MN517746
23316 Finland Picea abies Hylastes brunneus MN516703 MN517647 JF279990 MN517713 MN517747
L. cucullatum 1140=1141 H 218.83 Norway Picea abies Ips typographus AJ538335 MN517619 JF280000 MN517685 MN517724
1871 Japan Pinus jezoensis Ips typographus MN516704 MN517620 JF280001 MN517686 MN517725
5022 Austria Picea abies Ips typographus MN516705 MN517621 JF280002 MN517687 MN517726
23123 128299 Russia Picea abies Ips typographus MN516706 MN517622 JF280003 MN517688 JF280042
23190 Russia Pinus sylvestris Ips typographus MN516707 MN517623 JF280005 MN517689 JF280043
27983 Russia Picea abies Dryocoetus autographus MN516708 MN517624 MN517658 MN517690 MN517727
27984 Russia Picea abies Dryocoetus autographus MN516709 MN517625 MN517659 MN517691 MN517728
36623 Russia Picea abies Ips typographus MN516710 MN517626 MN517660 MN517692 MN517729
L. davidsonii 790 H Canada Pseudotsuga menziesii – MN516711 MN517627 MN517661 MN517693 MN517730
3094 Canada Picea sp. unknown bark beetle MN516712 MN517628 MN517662 MN517694 MN517731
3095 Canada Picea sp. unknown bark beetle MN516713 MN517629 MN517663 MN517695 MN517732
L. erubescens 40672 H 278.54 Sweden Pinus sylvestris – MN516714 MN517656 MN517683 MN517722 MN517756
L. francke-grosmanniae 445 H 356.77 Germany Quercus sp. Hylecoetus dermestoides MN516715 MN517618 MN517657 MN517684 MN517723
L. olivaceum 23348 128836 Finland Picea abies Ips typographus MN516717 MN517630 MN517664 MN517696 JF280049
23350 128837 Finland Picea abies Ips typographus MN516718 MN517631 MN517665 MN517697 JF280050
28090 Russia Pinus sylvestris Ips typographus MN516719 MN517632 MN517666 MN517698 MN517733
31059 H 138.51 Sweden Pinus sylvestris – MN516716 MN517633 JF279997 MN517699 MN517734
31060 152.54 Sweden – – MN516720 MN517634 JF279998 MN517700 MN517735
L. olivaceapini 63 503.86 USA – – MN516721 MN517635 MN517667 MN517701 MN517736
116 E 504.86 USA – – MN516722 MN517636 MN517668 MN517702 MN517737
L. pseudoalbum 40671 H 276.54 Sweden Pinus sylvestris Tomicus piniperda MN516723 MN517655 MN517682 MN517721 MN517755
L. rhizoidum 22809 H 136512 Spain Pinus radiata Hylastes ater MN516724 MN517648 MN517675 MN517714 MN517748
22810 P 136513 Spain Pinus radiata Hylastes attenuatus MN516725 MN517649 MN517676 MN517715 MN517749
22811 Spain Pinus radiata Ips sexdentatus MN516726 MN517650 MN517677 MN517716 MN517750
22812 Spain Pinus radiata Hylurgops palliatus MN516727 MN517651 MN517678 MN517717 MN517751
L. sagmatosporum 34135 E 113452 Canada Pinus strobus – MN516728 MN517637 MN517669 MN517703 MN517738
L. sylvestris 23300 P 128833 Finland Picea abies Ips typographus MN516729 MN517639 JF279996 MN517705 MN517740
34140 T 136511 Poland Pinus sylvestris – MN516730 MN517640 MN517671 MN517706 MN517741
L. vescum 34186 H 800.73 USA Picea engelmannii Ips pilifrons, Dendroctonus engelmanni MN516731 MN517638 MN517670 MN517704 MN517739
L. xiningense 38891 H 136509 China Picea crassifolia Polygraphus poligraphus MN516732 MN517652 MN517679 MN517718 MN517752
39237 P 136510 China Picea crassifolia Polygraphus poligraphus MN516733 MN517653 MN517680 MN517719 MN517753
39238 China Picea crassifolia Polygraphus poligraphus MN516734 MN517654 MN517681 MN517720 MN517754

1 Bold type = new species in the present study. 2 CMW = Culture Collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa; CBS = Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands. H = ex-holotype; E = ex-epitype; P = ex-paratype 3 ITS2 = the internal transcribed spacer 2 region of the nuclear ribosomal DNA gene; LSU = the 28S large subunit of the nrDNA gene; ACT= Actin; TUB = Beta-tubulin; CAL = Calmodulin; TEF-1α = Translation elongation factor 1-alpha; Bold type = Genbank accession numbers of sequences obtained in the present study.

DNA extraction, PCR and sequencing

DNA extractions were done as described by Yin et al. (2015). For sequencing and phylogenetic analyses, five loci were amplified: internal transcribed spacer 2 and large subunit (ITS2-LSU), actin (ACT), beta tubulin (TUB), calmodulin (CAL) and translation elongation factor-1 alpha (TEF-1α). Primers used were: ITS3 and LR3 (White et al. 1990) for ITS2-LSU, Lepact-F and Lepact-R (Lim et al. 2004) for ACT, T10 (O’Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995) for TUB, CL2F and CL2R (Duong et al. 2012) for CAL, EF2-F (Marincowitz et al. 2015) and EF2-R (Jacobs et al. 2004) for TEF-1α.

PCR reactions were conducted in 25 μL reaction mixtures containing 5 μL of Mytaq buffer (including MgCl2, dNTPs and reaction buffer), 0.5 μL of Mytaq polymerase (Bioline, USA), 0.5 μL of each primer (10 μM), and 16.5 μL of PCR grade water. PCR conditions for these five gene regions followed the protocols described by Yin et al. (2015). PCR products were purified with Sephadex G-50 columns (6%).

PCR products were sequenced with the same primers used for PCR, together with the Big Dye Terminator 3.1 cycle sequencing premix kit (Applied Biosystems, Foster City, California, USA). BigDye PCRs were conducted in 12 μL: sequencing Buffer 4.0 µL, Big Dye 1.0 µL, PCR Grade Water 4.0 µL, primer 1.0 µL, PCR product 2.0 µL; PCR conditions were: 1 min at 96 °C; 25 cycles of 10 sec at 96 °C, 5 sec at 50 °C, and 1min at 60 °C; and finally held at 12 °C. BigDye PCR products were also cleaned up with Sephadex. Sequence analyses were done on the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, California, USA). Consensus sequences were generated from forward and reverse sequences in the CLC Main Workbench 6.0 (CLC Bio, Aarhus, Denmark).

Phylogenetic analyses

Five sequence datasets were analyzed. The ITS2-LSU sequences of the ex-type isolate of every species in the L. olivaceum complex (Table 1) were compared with sequences of other known species in Leptographium obtained from GenBank to show the placement of the complex within the genus. Sequences of Fragosphaeria purpurea and F. reniformis were used to represent the outgroup taxa. Four protein coding gene regions (ACT, TUB, CAL, and TEF-1α) were sequenced (Table 1) for 39 isolates (Table 1) in order to delineate closely related species in the L. olivaceum complex. Sequences for L. procerum and L. profanum from the study of Yin et al. (2015) were selected to represent the outgroup taxa for the four protein-coding gene regions as well as in the combined dataset.

Alignments of loci were conducted in MAFFT 7.0 online (Katoh and Standley 2013), then checked manually in MEGA X (Kumar et al. 2018) and compared with the gene maps (Yin et al. 2015) to ensure that introns and exons were aligned appropriately. Three methods were used for phylogenetic analyses including Maximum parsimony (MP), Maximum Likelihood (ML), and Bayesian Inference (BI). A partition homogeneity test was conducted in PAUP* 4.0b10 (Swofford 2002) to consider the congruence of the four protein-coding gene regions before analyses of the combined dataset. The most important parameters used in phylogenetic analyses and statistical values related to all datasets analyzed are presented in Table 2.

MP analyses were executed in PAUP* 4.0b10 (Swofford 2002) with heuristic searches of 1000 replicates and tree bisection and reconnection (TBR) branch swapping options. Gaps were treated as the fifth base. Bootstrap analysis (1000 pseudo replicates) was performed to determine the confidence levels of the branch nodes. Tree length (TL), consistency Index (CI), retention Index (RI), Homoplasy Index (HI), and Rescaled Consistency Index (RC) were recorded after generating the trees.

The best substitution models (Table 2) for the two likelihood methods (ML and BI analyses) were selected congruously in jModelTest 2.1.1 (Pasoda 2008). MEGA X (Kumar et al. 2018) was used for ML analyses with Nearest-Neighbor-Interchange (NNI) branch swapping option. Node support values were determined using analysis of 1000 bootstrap pseudo replicates.

Table 2.
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Parameters used and statistical values related to all phylogenetic analyses in the present study.

ITS2-LSU ACT βT CAL TEF-1α Combined
Alignments Number of taxa 59 41 41 41 41 41
Total 603 809 288 579 781 2457
Constant 456 622 209 435 479 1785
Uninformative 46 20 8 22 45 95
Informative 101 127 71 122 257 577
MP Number of trees 396 13 4 15 10 12
Tree length 289 276 154 404 619 1486
CI 0.740 0.812 0.786 0.884 0.837 0.821
RI 0.934 0.935 0.933 0.956 0.941 0.935
RC 0.691 0.759 0.733 0.845 0.787 0.767
HI 0.259 0.188 0.214 0.116 0.163 0.179
Model tests Selected Models GTR+I+G HKY+I+G HKY+G HKY+I HKY+G HKY+I +G
ML P-inv 0.378 0.527 – 0.623 – 0.441
Gamma 0.257 0.287 0.179 – 0.618 0.712
BI Burn-in 100 300 300 300 300 300

MP = maximum parsimony, ML = maximum likelihood, BI = Bayesian inference, Uninformative = Number of parsimony-uninformative characters, Informative = Number of parsimony-informative characters, CI = consistency index, RI = retention index, RC = rescaled consistency index, HI = homoplasy index, Subst. model = substitution models used in phylogenetic analyses, P-inv = proportion of invariable sites, Gamma = Gamma distribution shape parameter.

For BI analyses, the Markov Chain Monte Carlo (MCMC) method was used in MrBayes 3.2 (Ronquist et al. 2012). Four MCMC chains were simultaneously run from a random starting tree for five million generations. Trees were sampled every 100 generations. Burn-in values were determined in Tracer v1.7 (Rambaut et al. 2018). Trees sampled in the burn-in phase were discarded and posterior probabilities were calculated from all the remaining trees.

Morphology and growth studies

In order to describe their morphology, isolates of new species were inoculated on to 2% water agar (WA, 20 g Difco agar and 1000 ml deionized water) amended with sterilized pine twigs (Pinus pinaster) and examined microscopically as described by Yin et al. (2015). Culture characteristics were recorded on Oatmeal agar (OA, 30 g oatmeal, 20 g Difco Bacto malt extract, from Becton, Dickinson and Company, and 1000ml deionized water) incubated at 25 °C for 10–14 days. Color descriptions were defined using the charts of Rayner (1970). Growth studies were conducted on 2 % Malt extract agar (MEA) following the procedure described by Yin et al. (2015).

Results

Phylogenetic analyses

The phylogenetic trees arising from the analyses of the ITS2-LSU data for Leptographium s.l. showed the L. olivaceum complex grouping between the L. galeiformis and L. procerum complexes with strong statistical support (Fig. 1). Within the complex, the ITS2-LSU sequences could not distinguish between some of the species, e.g. between L. rhizoidum and L. sagmatosporum; L. davidsonii and L. vescum; L. conplurium, L. pseudoalbum and L. erubescens. Leptographium francke-grosmanniae grouped peripheral to other species in the complex, but remained part of a strongly supported lineage including all the species under consideration.

Figure 1. 

Left side: ML tree of the genus Leptographium generated from the ITS2-LSU DNA sequence data. Sequences generated from this study are printed in bold type. Bold branches indicate posterior probabilities values ≥0.95. Bootstrap values ≥75% are recorded at nodes as ML/MP. * Bootstrap values <75%. Scale bar represents 5 nucleotide substitutions per 100 nucleotides. Right side: ML trees of the L. olivaceum complex generated from the DNA sequences of combined four protein-coding gene regions, including ACT, CAL, TEF-1α, and TUB. Bold branches indicate posterior probabilities values ≥0.95. Bootstrap values ≥75% are recorded at nodes as ML/MP. * Bootstrap values <75%. Scale bar represents 5 nucleotide substitutions per 100 nucleotides.

The ACT data matrix included part of exon 5 (sites 1–678), intron 5 (sites 679–785) and part of exon 6 (sites 786–809). The intron/exon composition of this gene region was congruent with that of the L. procerum complexes (Yin et al. 2015). Analyses of this gene region (Fig. 2) separated all known species and revealed six new taxa in the complex.

Figure 2. 

ML trees of the L. olivaceum complex generated from DNA sequences of four protein-coding gene regions. Bold branches indicate posterior probabilities values ≥0.95. Bootstrap values ≥75% are recorded at nodes as ML/MP. * Bootstrap values <75%. Scale bar represents nucleotide substitutions.

The TUB dataset included part of exon 4 (sites 1–41), intron 4 (sites 42–113), exon 5 (114–168) and part of exon 6 (sites 169–288). Intron 5 was lacking in the L. olivaceum complex, corresponding with most other species complexes in Leptographium s.l. (De Beer and Wingfield 2013). In the resulting phylogenies (Fig. 2), most known species and all new taxa could be separated, apart from the L. davidsonii and L. vescum isolates that formed a single clade.

The aligned DNA sequences for the CAL gene region included exon 3 (sites 1–16), intron 3 (sites 17–165), exon 4 (sites 166–291), intron 4 (sites 292–451), exon 5 (452–526), and part of exon 6 (sites 527–579). The intron/exon arrangement corresponded with that of the L. clavigerum and L. procerum complexes (Yin et al. 2015), with intron 5 lacking in this complex. Phylogenetic analyses of the CAL dataset (Fig. 2) recovered all currently accepted species in the complex.

The TEF-1α gene region used in phylogenetic analyses, included part of exon 3 (sites 1–9), intron 3 (sites 10–461), exon 4 (462–599), intron 4 (600–686), and part of exon 5 (687–781). Intron 4 of the TEF-1α gene was present in the L. olivaceum complex as is also true for the L. procerum, L. galeiformis, L. wageneri and L. serpens complexes, while it is absent in several other species complexes in Leptographium s. l. (De Beer and Wingfield 2013, Yin et al. 2015). Analysis of the TEF-1α dataset (Fig. 2) made it possible to separate all species in the complex.

The partition homogeneity test conducted on the combined data set for the four protein coding genes (ACT, TUB, CAL and TEF-1α) resulted in a P-value of 0.081, indicating that these regions could be combined. The MP, ML, and BI analyses generated were consistent with each other. Fourteen species with significant statistical support were defined in the L. olivaceum complex (Fig. 1), including eight known species (L. cucullatum, L. davidsonii, L. vescum, L. olivaceapini, L. erubescens, L. olivaceum, L. sagmatosporum, and L. francke-grosmanniae) and six new species from Europe and China.

Morphology and growth studies

Isolates of the six new species emerging from this study were similar in growth in culture, with colors initially hyaline, later turning pale yellowish or pale olivaceous. Mononematous synnemata were common in the cultures and hyphae were superficial on the agar. The droplets containing conidia were initially hyaline, becoming yellowish with age. Morphological differences among all these new species are discussed in the Notes sections provided with the new species descriptions in the Taxonomy section. A sexual state was induced only in isolates of L. sylvestris after incubation at 25 °C for three weeks.

Other than L. sylvestris that grew fastest at 30 °C, the optimal growth temperature for all isolates of the new species was 25 °C. None of the isolates of the new species grew at 5 °C or 35 °C, only L. rhizoidum was able to grow (2.5 mm/d) at 35 °C.

Taxonomy

Sequence data for 39 isolates included in the present study revealed 14 taxa in the L. olivaceum complex. One of these species, L. erubescens, was previously treated as a synonym of L. cucullatum but our data distinguished clearly between the two species. A new combination is thus provided for L. erubescens. Lectotypes and epitypes are designated here for L. olivaceum, L. sagmatosporum and L. erubescens. The remaining six taxa in the complex represented novel species and descriptions are provided for them.

Leptographium breviuscapum M.L. Yin, Z.W. de Beer & M.J. Wingf., sp. nov.

MycoBank No: 823576
Fig. 3

Etymology

The epithet (brevius-, short, and -scapum, branch) refers to very short conidiophores.

Type

CHINA, Qinghai province, from Picea crassifolia infested with Polygraphus poligraphus, Aug. 2010, M.L. Yin & X.D. Zhou, (PREM 60914 holotype, ex-holotype cultures CBS 136507 = CMW 38888); Qinghai province, from Picea crassifolia infested with P. poligraphus, Aug. 2010, M.L. Yin & X.D. Zhou, (PREM 60915 paratype, ex-paratype cultures: CBS 136508 = CMW 38889).

Description

Sexual state not observed. Conidiophores occasionally observed on wood of WA, macronematous, synnematous, short, wide at the stipe, light brown to yellowish, expanding branches at the apex, 150–230 μm in length including conidiogenous apparatus, 20–25 μm wide at base, 40–45 μm wide at apex, 100–150 μm wide at conidiogenous apparatus. Conidiogenous cells discrete, hyaline, cylindrical, percurrent proliferation, (8–)9–13(–15) × 1.8–2.5 μm. Conidia hyaline, one-celled, smooth, ellipsoidal, (3.7–)4–4.5(–5) × 2.5–3 μm. Culture characteristics: Colonies on OA, hyaline at first, later becoming light yellowish in the center, mycelium superficial on agar. Mostly mycelium observed in culture, synnemata sparse. Optimal temperature for growth 25 °C, growth reduced at 10 °C and 30 °C, no growth at 35 °C.

Figure 3. 

Leptographium breviuscapum sp. nov. (CMW 38888) a fourteen-days old culture on OA with black background b synnematous asexual state on wood tissue on WA c–d conidiophore e conidiogenous cells f conidia. Scale bars: 100 μm (b), 25 μm (c), 25 μm (d), 10 μm (e), 5 μm (f).

Host tree

Picea crassifolia.

Insect vector

Polygraphus poligraphus.

Distribution

Qinghai, China.

Note: The asexual state of L. breviuscapum has very short conidiophores making it very easy to distinguish from that of other species in the complex.

Additional material examined

Qinghai province, from Picea crassifolia infested with Polygraphus poligraphus, Aug. 2010, M.L. Yin & X.D. Zhou, (culture: CMW 38890). Yunnan province, from Pinus yunnanensis infested with Tomicus yunnanense, Sep. 2017, M.L. Yin, (culture: SCAU-475). Yunnan province, from Pinus yunnanensis infested with Tomicus yunnanense, Sep. 2017, M.L. Yin, (culture: SCAU-478).

Leptographium conplurium M.L. Yin, Z.W. de Beer & M.J. Wingf., sp. nov.

MycoBank No: 823572
Fig. 4

Etymology

The epithet refers to synnemata produced abundantly in culture.

Type

FINLAND, Ilomantsi, from Picea abies infested with Dryocoetes autographus, Aug. 2005, Z.W. de Beer, (PREM 60918-holotype, ex-holotype cultures: CBS 128923 = CMW 23315); Ilomantsi, from P. abies infested with D. autographus, Aug. 2005, Z.W. de Beer, (PREM 60919-paratype, ex-paratype cultures: CBS 128834 = CMW 23289).

Description

Sexual state not observed. Conidiophores macronematous, synnematous, 300–700 μm including conidiogenous apparatus, synnemata occasionally swollen at the base, frequently swollen at the stipe, brown to black, expanding branches at the apex, (25–)40–50(–80) μm in width, abundantly produced in culture. Conidiogenous cells discrete, terminal, hyaline, cylindrical, (8–)12–17(–20) × 1.5–2.3 μm. Conidia hyaline, one-celled, ellipsoidal to cylindrical, (3.9–)4.3–4.9(–6.3) × 1.9–2.5 μm. Culture characteristics: colonies on OA, hyaline at first, later becoming light yellowish in the center, concentric rings present, hyphae hyaline, appressed and immersed. Optimal growth temperature is 25 °C with radial growth rate 2.5 (± 0.5) mm/d, growth reduced at 10 °C and 30 °C, no growth at 35 °C.

Figure 4. 

Leptographium conplurium sp. nov. (CMW 23315). a fourteen-days old culture on OA with black background; b. synnematous asexual state on wood tissue on WA c conidiophore d conidiogenous apparatus e conidiogenous cells f conidia. Scale bars: 200 μm (b), 50 μm (c), 20 μm (d), 10 μm (e), 5 μm (f).

Host tree

Picea abies.

Insect vectors

Dryocoetes autographus, Hylastes brunneus.

Distribution

Finland.

Notes

All isolates of this species were initially recognized as a cryptic species closely related to L. cucullatum and L. olivaceapini by Linnakoski et al. (2012). Our results confirmed that they represent an undescribed taxon.

Additional material examined

FINLAND, Ilomantsi, from Picea abies infested with Dryocoetes autographus, Aug. 2005, Z.W. de Beer, (culture: CMW 23295); Ilomantsi, from P. abies infested with Hylastes brunneus, Aug. 2005, Z.W. de Beer, (culture: CMW 23316).

Leptographium cucullatum (H. Solheim) M.L. Yin, Z.W. de Beer & M.J. Wingf., comb. nov.

MycoBank No: 831546

≡ Ophiostoma cucullatum H. Solheim, Nord. J. Bot. 6: 202 (1986). (Basionym)

≡ Grosmannia cucullata (H. Solheim) Zipfel, Z.W. de Beer & M.J. Wingf., Zipfel et al. (2006) Stud. Mycol. 55: 90.

Type

NORWAY, Vestfold, Lardal, from Ips typographus caught when leaving a log of Picea abies, 20 Aug 1981, H. Solheim, (CBS H-15306 and CBS H-3560-holotype, ex-holotype cultures: CMW 1140 = CBS 218.83 = 81-83/16).

Descriptions

Solheim (1986, pp 202–203, fig. 2); Wingfield et al. (1989, pp 92–95, figs 1–10); Yamaoka et al. (1997, pp 1220–1221 figs 22–26); Harrington et al. (2001, pp 128–129, figs 41, 44).

Host trees

Picea abies, Picea jezoensis, Pinus sylvestris.

Insect vectors

Dryocoetus autographus, Ips typographus, Ips typographus japonicus.

Distributions

Europe (Austria, Norway, Poland, Russia), Japan

Notes

Harrington et al. (2001) suggested that Phialographium erubescens represented the asexual state of L. cucullatum. Comprehensive data from the present study distinguish between the two species. See details under L. erubescens.

Additional material examined

AUSTRIA, Tyrol, Ehrwald, from I. typographus in Picea abies, July 1997, T. Kirisits, CMW 5022; JAPAN, Hokkaido, Furano, from an adult of Ips typographus japonicus in Picea jezoensis, 31 July 1991, Y. Yamaoka, CMW 1871 = JCM 8816; RUSSIA, Ohtama, from I. typographus in P. abies, June 2004, J. Ahtiainen, CMW 23123 = CBS 128299;RUSSIA, Lisino-Corpus,from I. typographus in Pinus sylvestris, R. Linnakoski, CMW 23190; RUSSIA, Kivennapa, Lintula, from Dryocoetus autographus in P. abies, Oct 2007, R. Linnakoski, CMW 27983, CMW 27984; RUSSIA, Karelia, from I. typographus in P. abies, H. Roininen, CMW 36623.

Leptographium davidsonii (Olchow. & J. Reid) M.L. Yin, Z.W. de Beer & M.J. Wingf., comb. nov.

MycoBank No: 831547

≡ Ceratocystis davidsonii (Olchow. & J. Reid), Can. J. Bot. 52: 1698 (1974). (Basionym)

≡ Ophiostoma davidsonii (Olchow. & J. Reid) H. Solheim, Nord. J. Bot. 6: 203 (1986).

≡ Grosmannia davidsonii (Olchow. & J. Reid) Zipfel, Z.W. de Beer & M.J. Wingf., Zipfel et al., Stud. Mycol. 55: 90 (2006).

Type

CANADA, British Columbia, Seymour Arm, from Pseudotsuga menziesii, 1971, J. Reid, (WIN (M) 71-30-holotype, ex-holotype cultures: CMW 790 = IMI 176524 = JCM 7867).

Descriptions

Olchowecki & Reid (1974, pp 1698–1699, figs 230–238); Upadhyay (1981, pp 42–43, figs 58–62); Mouton et al. (1993, pp 376–377, figs 15–18); Ohtaka et al. (2002, pp 154–156, figs 6–10).

Host trees

Abies veitchii, Picea sp, Pseudotsuga menziesii.

Insect vector

Dryocoetes hectographus.

Distribution

USA, Japan.

Notes

The orange section shaped to hemispherical ascospores makes this species distinct from others in the complex (Ohtaka et al. 2002). This fungus was also reported associated with Dryocoetes hectographus on Abies veitchii in Japan based on morphology (Ohtaka et al. 2002), but the identity of the Japanese isolates needs to be verified with DNA sequences.

Additional material examined

CANADA, British Columbia, Lake Louise, from small Scolytinae sp. in Picea sp. Aug 1994, M. J. Wingfield, (cultures: CMW 3094, CMW 3095).

Leptographium erubescens (Math.-Käärik) M.L. Yin, Z.W. de Beer & M.J. Wingf., comb. nov.

MycoBank No: 823577

≡ Graphium erubescens Math.-Käärik, Medd. Skogs for skninginst. 43: 62 (1953). (Basionym)

≡ Pesotum erubescens (Math.-Käärik) G. Okada, Stud. Mycol. 45: 184 (2000).

≡ Phialographium erubescens (Math.-Käärik) T.C. Harr. & McNew, Mycologia 93: 129 (2001).

Type

SWEDEN, from pine poles and board, A. Mathiesen-Käärik, lectotype designated here, represented by line drawings (fig. 8b, p. 58; fig. 9d–f, p. 61) from Mathiesen-Käärik (1953), MBT 379456; Uppland, Skutskär, from piled timber of Pinus sylvestris, 1952, A. Mathiesen-Käärik, (Isotype CBS H-7193, CBS H-7194, ex-type cultures: CMW 40672 = CBS 278.54 = JCM 9747 = No. Sk 13-52).

Descriptions

Mathiesen-Käärik (1953, p.62, figs8b, 9f–d); Harrington et al. (2001, pp 128–129, figs 42, 43, 45).

Host tree

Pinus sylvestris.

Insect vector

unknown.

Distribution

Sweden.

Notes

This species was first described by Mathiesen-Käärik (1953) from pine timber in Sweden. No specimen numbers and very little detail (e.g. no host locality or collection dates) were provided in the protologue. Furthermore, no specimen number and little detail are listed under this species name in the herbarium of the Museum of Evolution, Uppsala, which incorporated Mathiesen-Käärik’s collection. However, in 1954 she deposited an isolate (No. Sk 13-52) in the CBS labeled as L. erubescens. Two dried specimens (CBS H-7193, CBS H-7194) are linked to this isolate and these are labeled as isotypes. It is reasonable to assume that this isolate represents the original material, but there is no conclusive evidence that this is true. We have thus designated the line drawings from the protologue (Mathiesen-Käärik 1953) as the lectotype.

Harrington et al., (2001) suggested that Graphium erubescens (as Phialographium erubescens) represented the asexual state of L. cucullatum (as O. cucullatum) based on ITS sequences. However, based on sequences produced in the present study, the ex-type culture of L. erubescens differs from that of L. cucullatum in 1bp in ITS2-LSU, 17 bp in ACT, 17 bp in BT, 30 bp in CAL, and 48 bp in TEF-1α. We have thus treated these species as distinct and have provided a new combination for L. erubescens.

Leptographium francke-grosmanniae (R.W. Davidson) K. Jacobs & M.J. Wingf., Leptographium species: p. 99 (2001)

MycoBank No: MB375135

≡ Ceratocystis francke-grosmanniae R.W. Davidson, Mycologia 63: 6 (1971). (Basionym)

≡ Ophiostoma francke-grosmanniae (R.W. Davidson) De Hoog & R.J. Scheff., Mycologia 76: 297 (1984).

≡ Grosmannia francke-grosmanniae (R.W. Davidson) Zipfel, Z.W. de Beer & M.J. Wingf., Stud. Mycol. 55: 90 (2006).

Type

GERMANY, Reinbeck near Hamburg, from Quercus sp. associated with Hylecoetus dermestoides, May 1967, H. Francke-Grosmann, (holotype BPI 595654, ex-holotype cultures: RWD 828 = ATCC 22061 = CBS 356.77 = CMW 445).

Descriptions

Davidson (1971, pp 6–7, figs 1, 10, 11, 17); Upadhyay (1981, p. 45, figs 73–78); Mouton et al. (1992, figs 1–11); Wingfield (1993, p. 48, figs 6–7); Jacobs and Wingfield (2001, pp 99–102, figs 73–75).

Host tree

Quercus sp.

Insect vector

Hylecoetus dermestoides.

Distribution

Germany.

Notes

Leptographium francke-grosmanniae groups peripheral to other species in the L. olivaceum complex (Figs 1–3). Morphologically, the ascospores are almost cylindrical and its ascomatal necks correspond with other species in the complex. But L. francke-grosmanniae produces mononematous conidiophores, in contrast to the synnemata produced by the other species, which also explains why it is the only species in the complex previously treated in Leptographium. The mode of conidiogenesis of L. francke-grosmanniae (Mouton et al. 1992) appears similar to that of other species where the conidiogenous cells that appear phialidic under a light microscope arise from percurrent proliferation (Wingfield et al. 1989, Wingfield et al. 1991, Mouton et al. 1993). However, the apices of the apparent “phialides” are substantially more flared than those of other species in the complex and they could be more different than assumed by Mouton et al. (1993). Leptographium francke-grosmanniae is also unusual in the L. olivaceum complex in having an angiosperm host.

Leptographium francke-grosmanniae was originally described as Ceratocystis francke-grosmanniae from larval galleries of Hylecoetus dermestoides on Quercus sp. in Germany (Davidson 1971). De Beer and Wingfield (2013) showed that sequences for this species produced in different studies were inconsistent. Based on comparisons of the ITS2 region, the sequences of ex-holotype generated in the present study are consistent with those produced by Mullineux and Hausner (2009) for ATCC 22061 and Hamelin et al. (unpublished) for CBS 356.77, but differ substantially from sequences produced by Jacobs et al. (2001b). In the LSU gene region, our sequences are identical to those of Hausner et al. (2000), but they differed from that of Jacobs et al. (2001a, b) for CMW 445.In the β-tubulin gene region, the sequence of CMW 445 in the present study was consistent with that provided by Kim et al. (2004) for CMW 445 and Hamelin et al. (unpublished sequence in GenBank) for CBS 356.77. We thus suggest that the two sequences for L. francke-grosmanniae produced by Jacobs et al. (2001a, b) are incorrect. Sequences of another isolate from the USA (CMW 2975), previously identified as L. francke-grosmanniae (Zipfel et al. 2006), differ substantially from the ex-holotype culture. Thus, this isolate (CMW 2975) does not represent L. francke-grosmanniae, and its taxonomic placement needs reconsideration.

Leptographium olivaceum (Math.-Käärik) M.L. Yin, Z.W. de Beer & M.J. Wingf., comb. nov.

MycoBank No: 831548

≡ Ophiostoma olivaceum Math.-Käärik, Svensk. Bot. Tidskr. 45: 212 (1951). (Basionym)

≡ Ceratocystis olivacea (Math.-Käärik) J. Hunt, Lloydia 19: 29 (1956).

≡ Grosmannia olivacea (Math.-Käärik) Zipfel, Z.W. de Beer & M.J. Wingf., Zipfel et al., Stud. Mycol. 55: 91 (2006).

Type

SWEDEN, Hällnäs, Västerbotten, from the galleries of Acanthocinus aedilis in pine wood, A. Mathiesen-Käärik, lectotype designated here, represented by line drawings (fig. 2a–g, p. 213) from Mathiesen-Käärik (1951), MBT 379459; from dead wood of Pinus sylvestris, Jan 1949, A. Mathiesen-Käärik, (ex-type cultures: CMW 31059 = CBS 138.51, MBT 2063).

Descriptions

Mathiesen-Käärik (1950, p. 298); Mathiesen-Käärik (1951, pp 212–215, fig. 2); Hunt (1956, pp 29–30); Griffin (1968, pp 707–708, figs 49–52, 82); Olchowecki and Reid (1974, pp 1699–1700, Pl. XIII fig. 262); Upadhyay (1981, pp 52–54, figs 116–121); Mouton et al. (1993, pp 376–377, figs 19–22).

Host trees

Betula papyrifera, Picea abies, Picea mariana, Pinus sylvestris.

Insect vectors

Acanthocinus aedilis, Dendroctonus rufipennis, Ips typographus, Polygraphus rufipennis.

Distributions

Canada, Finland, Russia, Sweden, USA.

Notes

This species was first described invalidly (no Latin diagnosis) from Pinus sylvestris infested by a longhorn beetle Acanthocinus aedilis in Sweden (Mathiesen-Käärik 1950). Mathiesen-Käärik (1951) then validated the name with a more detailed description accompanied by a Latin diagnosis. In the original descriptions of L. olivaceum by Mathiesen-Käärik (1950, 1951), the host tree, beetle and location of the collection was noted, but no mention was made of a specimen. The herbarium specimens of Mathiesen-Käärik were initially curated in the herbarium of the Statens Skogsforsknings institut, Experimentalfältet, Sweden. The collection was later incorporated into the herbarium of the Museum of Evolution, Uppsala. Only one herbarium specimen (UPS:BOT:F-130986) of L. olivaceum, collected from the same host, beetle and location by T. Hedquist, is available from that collection. However, an isolate of L. olivaceum (No. 297-49 = CBS 138.51), collected in 1949, also from the original host and location, was deposited in the CBS by Mathiesen-Käärik in 1951. Although we were not able to confirm that this isolate was from the original collection, it was treated as the ex-type culture of the species in previous studies (Duong et al. 2012, Linnakoski et al. 2012, De Beer and Wingfield 2013). In view of the absence of concrete evidence that this isolate represents the original material, we have designated the line drawings from the protologue (Mathiesen-Käärik 1951) as lectotype.

More recently, it was reported from Picea abies and Pinus sylvestris infested by Ips typographus and Dryocoetes autographus in Finland and Russia, in a study where the identities were confirmed using DNA sequence analyses (Linnakoski et al. 2012). Griffin (1968) reduced L. vescum to synonymy with L. olivaceum, but data from the present study confirmed that these two species are phylogenetically distinct.

Additional material examined

FINLAND, Jouhteninen, from Ips typographus in Picea abies, July 2005, Z.W. de Beer, (cultures: CMW 23348 = CBS 128836, CMW 23350 = CBS 128837). RUSSIA, Uuksujärvi, from I. typographus in Pinus sylvestris, Oct 2007, R. Linnakoski, (culture CMW 28090). SWEDEN, Oct 1954, A. Mathiesen-Käärik, (cultures: CMW 31060 = CBS 152.54).

Leptographium olivaceapini (R.W. Davidson) M.L. Yin, Z.W. de Beer & M.J. Wingf., comb. nov.

MycoBank No: 831549

≡ Ceratocystis olivaceapini R.W. Davidson, Mycologia 63: 7 (1971). (Basionym)

≡ Ophiostoma olivaceapini (R.W. Davidson) K.A. Seifert & G. Okada, In Okada et al., Can. J. Bot. 76: 1504 (1998).

≡ Grosmannia olivaceapini (R.W. Davidson) Z.W. de Beer, R. Linnakoski & M.J. Wingf., In Linnakoski et al., Antonie van Leeuwenhoek 102: 375–399 (2012).

Type

USA, New Mexico, Santa Fe, from Pinus ponderosa tree infested Dendroctonus sp. and other bark beetles, 10 July 1964, R.W. Davidson, (holotype BPI 595910 = RWD 548D; BPI 595914 = RWD 548D isotype); USA, Arizona, Flagstaff, from Pinus ponderosa infested with Dendroctonus sp., 24 July 1964, R.W. Davidson, (BPI 596223= RWD 581-D isotype); Arizona, Flagstaff, from P. ponderosa infested with Dendroctonus sp., 3 Oct 1986, T. Hinds, (epitype PREM 61051, designated here, ex-epitype cultures CBS 504.86 = CMW 116 = COLO 479, MBT 379458).

Descriptions

Davidson (1971, pp 7–10, figs 2, 12, 18); Upadhyay (1981, p. 54, figs 122–129); Mouton et al. (1993, pp 372–373, figs 1–4).

Host trees

Pinus ponderosa.

Insect vectors

Dendroctonus sp.

Distribution

USA.

Notes

No living culture associated with the holotype (BPI 595910) or isotype (BPI 595914) of L. olivaceapini exists. However, T. Hinds, a collaborator of R.W. Davidson and later curator of the RWD culture collection, provided an isolate (COLO 479) labeled as C. olivaceapini to M.J. Wingfield, who later deposited this in the CBS (CBS 504.86). The species name and origin provided by Hinds with the isolate corresponds to a second specimen mentioned by Davidson (1971, p. 10) in the protologue (RWD 581-D = BPI 596223). In our opinion, the isolate (COLO 479) most probably originated from the specimen (RWD 581-D). We could not confirm with certainty that BPI 296223 originated from RWD 581-D and thus designated a dried culture of COLO 479 as the epitype for L. olivaceapini.

Additional Material examined: USA, Arizona, Flagstaff, from P. ponderosa infested with Dendroctonus sp., 3 Oct 1986, T. Hinds, (PREM 61051, cultures CBS 504.86 = CMW 116 = COLO 479). Minnesota, Pinus resinosa, Nov 1986, M.J. Wingfield, (cultures: CBS 503.86 = CMW 63).

Leptographium pseudoalbum M.L. Yin, Z.W. de Beer and M.J. Wingf., sp. nov.

MycoBank No: 823571
Fig. 5

Etymology

The epithet refers to the previous, incorrect identification of the ex-holotype isolate of this species as Graphium album.

Type

SWEDEN, from Pinus sylvestris infested by Tomicus piniperda, 1953, Mathiesen-Käärik, (PREM 61050-holotype, ex-holotype cultures: CBS 276.54 = CMW 40671 = JCMW 9774 = C 1225).

Description

Sexual state not observed. Conidiophores macronematous, synnematous, 120–270 μm including conidiogenous apparatus, synnemata frequently swollen at base, frequently wider at stipe, expanding branches at apex, brown to hyaline, (11–)25–34(–40) μm in width. Conidiogenous cells discrete, terminal, percurrent and phialidic proliferation, hyaline, cylindrical, (9–)10–14(–18) × 1.8–2.8 μm. Conidia hyaline, one-celled, ellipsoidal to cylindrical, (3.5–)4.3–5.2(–6.5) × 2.4–3.3 μm. Cultural characteristics: Colonies on OA, hyaline at first, later becoming white and gray in the center, hyphae hyaline, appressed and immersed, aerial mycelium frequently present on wood tissue, phialographium-like asexual morph abundant. Optimal growth temperature on MEA:25 °C with radial growth rate 3.0 (± 0.5) mm/d, while growth slightly reduced at 10 °C and 30 °C, and no growth occurred at 35 °C.

Figure 5. 

Leptographium pseudoalbum sp. nov. (CBS 276.54) a fourteen-days old culture on OA with black background; b. synnematous asexual state on wood tissue on WA c–d conidiophore e conidiogenous cells f conidia. Scale bars: 200 μm (b), 25 μm (c), 25 μm (d), 10 μm (e), 5 μm (f).

Host

Pinus sylvestris.

Insect vector

Tomicus piniperda.

Distribution

Sweden.

Notes

This species was initially identified as Graphium album (Corda) Sacc. by Mathiesen-Käärik (1953). However, Okada et al. (2000) and Harrington et al. (2001) questioned the identification by Mathiesen-Käärik (1953) and showed that this isolate belonged in the Ophiostomatales and grouped close to L. erubescens. This study showed that Mathiesen-Käärik’s isolate representing an undescribed species in the L. olivaceum complex, for which we have provided the name L. pseudoalbum.

Leptographium rhizoidum M.L. Yin, Z.W. de Beer and M.J. Wingf., sp. nov.

MycoBank No: 823575
Fig. 6

Etymology

The epithet refers to the rhizoid-like structures at the synnematal bases.

Type

SPAIN, Morga, from Pinus radiata infested by Hylastes ater, July. 2004, P. Romon & X.D. Zhou, (PREM 60922-holotype, ex-holotype cultures: CBS 136512 = CMW 22809); Morga, from Pinus radiata infested by Hylastes attenuatus, July. 2004, P. Romon & X.D. Zhou, (PREM 60923-paratype, ex-paratype cultures: CBS 136513 = CMW 22810).

Description

Sexual state not observed. Conidiophores macronematous, synnematous, 200–350 μm including conidiogenous apparatus, synnemata frequently swollen at the base, frequently wider at the stipe, brown to light brown, expanding branches at the apex, (15–)35–45(–70) μm in width. Conidiogenous cells discrete, terminal, percurrent and phialidic proliferation, hyaline, cylindrical,(10–)14–17(–19) × 2–3 μm. Conidia hyaline, one-celled, cylindrical to obovoid, (5.1–)6.5–7.8(–10.5) × 2.1–3.5 μm. Cultural characteristics: Colonies on OA, hyaline at first, later becoming olivaceous in the center, hyphae hyaline, appressed and immersed, aerial mycelium frequently present on wood tissue, synnemata abundant in WA cultures, Optimal growth temperature on MEA is 25 °C with radial growth rate 6.0 (± 0.5) mm/d, growth slightly reduced at 10 °C and 35 °C.

Figure 6. 

Leptographium rhizoidum sp. nov. (CMW 22809). a fourteen-days old culture on OA with black background b synnematous asexual state on wood tissue on WA c conidiophore d conidiogenous apparatus e conidiogenous cells f conidia. Scale bars: 200 μm (b), 50 μm (c), 20 μm (d), 10 μm (e), 5 μm (f).

Host tree

Pinus radiata.

Insect vectors

Hylastes ater, H. attenuatus, Hylurgops palliatus, Ips sexdentatus.

Distribution

Spain.

Note: Isolates of L. rhizoidum from pine-infesting bark beetles in Spain were initially identified as L. olivaceum based on ITS sequences by Romon et al. (2007). Our data showed them to be distinct from that species. This species produced more abundant and longer rhizoids than others in the complex.

Other Material examined: SPAIN, Morga, from Pinus radiata infested by Ips sexdentatus, July. 2004, P. Romon & X.D. Zhou, (culture: CMW 22811); Morga, from P. radiata infested by Hylurgops palliatus, July. 2004, P. Romon & X.D. Zhou, (culture: CMW 22812).

Leptographium sagmatosporum (E.F. Wright & Cain) M.L. Yin, Z.W. de Beer & M.J. Wingf., comb. nov.

MycoBank No: 831550

≡ Ceratocystis sagmatospora E.F. Wright & Cain, Can. J. Bot. 39: 1226 (1961). (Basionym).

≡ Phialographium sagmatosporae H.P. Upadhyay and W.B. Kendr., Mycologia 66: 183 (1974).

≡ Ophiostoma sagmatosporum (E.F. Wright & Cain) H. Solheim, Nord. J. Bot. 6: 203 (1986).

≡ Graphium sagmatosporae (H.P. Upadhyay & W.B. Kendr.) M.J. Wingf. & W.B. Kendr., Mycol. Res. 95: 1332 (1991).

≡ Pesotum sagmatosporum (H.P. Upadhyay & W.B. Kendr.) G. Okada & K.A. Seifert, in Okada et al., Can. J. Bot. 76: 1504 (1998).

≡ Grosmannia sagmatospora (E.F. Wright & Cain) Zipfel, Z.W. de Beer & M.J. Wingf., In Zipfel et al. Stud. Mycol. 55: 91 (2006).

Type

CANADA, Ontario, Ontario, NE. of Mansfield, Dufferin Co., from Pinus resinosa, Nov. 8 1958, E.F. Wright &R.F. Cain, lectotype designated here, represented by line drawings (fig. 23, p. 1225, figs 24–33, p. 1228) from Wright and Cain (1961), MBT 379455; Ontario, Stittsville, 13 Lucas Lane, 4511.9 N 7558.8 W, from old bark beetle galleries in Pinus strobus, Sept. 2000, K. Jacobs, (epitype PREM 61054, designated here, ex-epitype cultures: CMW 34135 = CBS 113452, MBT 379454).

Descriptions

Wright and Cain (1961, pp 1226–1229, figs 23–33); Griffin (1968, pp 708, 712–713); Olchowecki and Reid (1974, p. 1701, Pl. XIII figs 254, 257); Upadhyay (1981, p. 60, figs 167–171).

Host trees

Pinus strobus, Picea mariana.

Insect vectors

unknown bark beetle species.

Distribution

Canada.

Notes

This species was originally described from bark beetle galleries and freshly cut surfaces of Picea mariana, Pinus resinosa and Pinus strobus in Canada (Wright and Cain 1961). The Royal Ontario Museum Fungarium (TRTC), Canada, informed the authors of this study that the holotype (TRTC 36427) of L. sagmatosporum was permanently lost. There is also no living culture available from the holotype. We have thus designated the line drawings in the protologue as the lectotype. An isolate (CMW 34135), also from pine in Ontario, identified as L. sagmatosporum based on morphology (K. Jacobs, unpublished) and used in previous studies to represent the species (Duong et al. 2012, Linnakoski et al. 2012, De Beer and Wingfield 2013), its dry specimen is designated here as the epitype.

Additional Material examined: CANADA, Ontario, NE. of Mansfield, Dufferin Co., from Pinus resinosa, Nov. 8 1958, E.F. Wright & R.F. Cain, TRTC 34600; NW. of Nobleton, York Co., from Pinus strobus, July 1 1957, E.F. Wright & R.F. Cain, TRTC 33034; Twp. West of 11 H, Challener Lake, Sudbury Dist., from Pinus strobus, June 20 1960, E.F. Wright & R.F. Cain, TRTC 36245, 36251, 36255, 36264, 36265;Twp. 5F, Aubinadong R., Algoma Dist, from Pinus strobus, June 17 1960, E.F. Wright & R.F. Cain, TRTC 36246; Twp. West of 11 H, Challener Lake, Sudbury Dist., from Picea mariana, June 20 1960, E.F. Wright & R.F. Cain, TRTC 36263.

Leptographium sylvestris M.L. Yin, Z.W. de Beer and M.J. Wingf., sp. nov.

MycoBank No: 823574
Fig. 7

Etymology

The epithet refers to the host species where the holotype was collected.

Type

POLAND, Chrosnica, from Pinus sylvestris, Jan. 2008, R. Jankowiak, (PREM 60920-holotype, ex-holotype cultures: CBS 136511 = CMW 34140). FINLAND, Jouhteninen, from Picea abies infested with Ips typographus, Aug. 2005, Z.W. de Beer, (PREM 60921-paratype, ex-paratype cultures: CBS 128833 = CMW 23300).

Description

Sexual state develop on wood on WA in 14–21 days. Perithecia superficial on wood and agar, base brown to black, globose, unornamented, 91–110 μm in diameter, necks dark brown, cylindrical, slightly curved, 200–480 μm long (including ostiolar hyphae), 26–32 μm wide at base, 15–21 μm wide at the tip. Ostiolar hyphae present, pale brown, straight, septate, numerous, divergent, tapering at the tip, up to 190 μm long. Asci not seen. Ascospores one-celled, hyaline, fusiform to orange section shaped in side view, ellipsoidal in face view, globose in end view, (4.0–)4.5–5.5(–5.8) × (2.5–)2.8–3.7(–3.9) μm including hyaline gelatinous sheath, 0.3–0.6 μm thick. Conidiophores macronematous, synnematous, swollen at the base, occasionally wider at the stipe, brown to light brown, expanding branches at the apex, 260–500 × 14–57 μm including conidiogenous apparatus. Conidiogenous cells discrete, hyaline, cylindrical, 2–3 per branch, percurrent proliferation, (10–)11–15(–18) × 1.5–2.5 μm. Conidia hyaline, obovate to clavate, (3.6–)4.5–4.9(–5.2) × (1.6–)1.7–1.9(–2.1) μm. Cultural characteristics: Colonies on OA, hyaline at first, later becoming dark yellowish in the center, mycelium appressed and immersed, Perithecia and Pesotum-like asexual morph co-occur in culture. Optimal growth temperature is 30 °C, radial growth rate 5.0 (± 0.5) mm/d, growth reduced at 10 °C, no growth at 35 °C.

Figure 7. 

Leptographium sylvestris sp. nov. (CMW 34140) a fourteen-days old culture on OA with black background b synnematous asexual state on wood tissue on WA c conidiophore d conidiogenous apparatus e conidiogenous cells f conidia g–h the sexual state on wood tissue on WA i ascoma j ostiolar hyphae k ascomatal base l ascospores. Scale bars: 100 μm (b), 50 μm (c), 25 μm (d), 10 μm (e), 5 μm (f), 100 μm (g), 100 μm (h), 50 μm (i), 25 μm (j), 20 μm (k), 5 μm (l).

Host trees

Pinus sylvestris, Picea abies.

Insect vector

Ips typographus.

Distributions

Poland, Finland.

Notes

The Finnish isolate (CMW 23300) was considered by Linnakoski et al. (2012) to be the same undescribed species as the isolates described above as L. conplurium. The addition of a newly obtained isolate from Poland in the present study, confirmed that the two isolates represented a distinct taxon, clearly separated from all other species in the complex. This is the only new species for which ascomata were obtained in culture. Single ascospore isolates of this species produced ascomata in culture, suggesting that the species is homothallic. The common characters of sexual states of species in this complex are having ascomata with sheath and ostiolar hyphae on the top of neck. This species differs from others by its fusiform to orange section shaped ascospores and slightly curved neck.

Leptographium vescum (R.W. Davidson) M.L. Yin, Z.W. de Beer & M.J. Wingf., comb. nov.

MycoBank No: 831551

≡ Ceratocystis vesca R.W. Davidson, Mycologia 50: 666. (1958) (Basionym)

≡ Ophiostoma vescum (R.W. Davidson) Hausner, J. Reid & Klassen. Can. J. Bot. 71: 1264. (1993)

≡ Grosmannia vesca (R.W. Davidson) Zipfel, Z.W. de Beer & M.J. Wingf., Zipfel et al., Stud. Mycol. 55: 92. (2006)

Type

USA, Colorado, Fort Collins, from Ips pilifrons and Dendroctonus engelmanni in Picea engelmannii, Jan. 31, 1956, F.F. Lombard & R.W. Davidson, (holotype BPI 595662 = FP 70807, ex-holotype cultures: ATCC 12968 = CBS 800.73 = CMW 34186).

Descriptions

Davidson (1958, p. 666); De Hoog and Scheffer (1984, p. 295, fig. 2); Samuels (1993, p. 16, fig. 1C–F).

Host tree

Picea engelmannii.

Insect vectors

Ips pilifrons, Dendroctonus engelmanni.

Distribution

USA.

Notes

The perithecia of L. vescum are smaller than in related species and ascospores are different in shape and size. This species was treated as a synonym of L. olivaceum by various authors (Griffin 1968, Olchowecki and Reid 1974, Upadhyay 1981). However, the sequences produced by Hausner et al. (1993, 2000), confirmed by our results, showed that the two species are distinct.

Leptographium xiningense M.L. Yin, Z.W. de Beer and M.J. Wingf., sp. nov.

MycoBank No: 823573
Fig. 8

Etymology

The epithet refers to the locality where the species was first collected.

Type

CHINA, Qinghai Province, from Picea crassifolia infested by Polygraphus poligraphus, Aug. 2010, M.L. Yin & X.D. Zhou, (PREM 60916-holotype, ex-holotype cultures CBS 136509 = CMW 38891); Qinghai Province, from Picea crassifolia infested by Polygraphus poligraphus, Aug. 2010, M.L. Yin, (PREM 60917-paratype, ex-paratype cultures CBS 136510 = CMW 39237).

Description

Sexual state not observed. Conidiophores macronematous, synnematous, 450–550 μm including conidiogenous apparatus, synnemata occasionally slightly swollen at the base, wider at the stipe, black to brown, expanding branches at the apex, light brown to hyaline, (25–)39–44(–50) μm in width. Conidiogenous cells discrete, terminal, percurrent and phialidic proliferation, hyaline, cylindrical, (11–)15–18(–19) × 2–3 μm. Conidia hyaline, one-celled, cylindrical to obovoid, (3.9–)4.2–4.5(–4.8) × 1.8–2.4 μm. Cultural characteristics: Colonies on OA, spore drops hyaline at first, later becoming light to dark yellowish in the center, hyphae hyaline, appressed and immersed, synnemata predominant, aerial mycelium occasionally present on wood tissue, Optimal growth temperature on MEA is 25 °C with radial growth rate 2.0 (± 0.5) mm/d, growth reduced at 10 °C, no growth at 30 °C.

Figure 8. 

Leptographium xiningense sp. nov. (CMW 38891) a fourteen-days old culture on OA with black background b synnematous asexual state on wood tissue on WA c conidiophore d conidiogenous apparatus e conidiogenous cells f conidia. Scale bars: 300 μm (b), 50 μm (c), 20 μm (d), 10 μm (e), 5 μm (f).

Host tree

Picea crassifolia.

Insect vector

Polygraphus poligraphus.

Distribution

China.

Note

This species groups closely with L. conplurium and L. erubescens, but can be distinguished by its dark conidial droplets. In addition, the synnematous conidiophores of this species were shorter, and its conidia were bigger than that of L. erubescens.

Additional material examined

CHINA, Qinghai Province, from Picea crassifolia infested by Polygraphus poligraphus, Aug. 2010, M.L. Yin & X.D. Zhou, (culture: CMW 39238). Chongqing, from Pinus armandii infested by Dendroctonus armandi, Nov. 2018, M.L. Yin, (culture: SCAU-530). Chongqing, from Pinus armandii infested by Dendroctonus armandi, Nov. 2018, M.L. Yin, (culture: SCAU-531).

Discussion

Among the five loci used in the phylogenetic analyses, ACT, CAL, and TEF-1α were able to distinguish among all species in the L. olivaceum complex. In contrast, TUB sequences could not distinguish between L. davidsonii and L. vescum. Although ITS2-LSU sequences provided reasonable resolution for species complexes at the genus level, this region could not be used to distinguish among closely related species. Of the five gene regions, TEF-1α had the most variable sites and this is consistent with the results of Yin et al. (2015) for the L. procerum complex. This also supports their suggestion that TEF-1α is suitable for use as a barcoding gene for accurate species identification in Leptographium.

In this study, we have clarified the previous confusion related to the ex-type isolate of L. francke-grosmanniae, and although our phylogenetic data placed it close to the complex, it grouped separated from all other species. This is consistent with its mononematous morphology that distinguishes it from all other species in the complex that produce synnematous asexual states. Furthermore, it is unique in that it does not come from the galleries of a conifer-infesting scolytine bark beetle like the other species, but from the large timberworm beetle, Hylecoetus dermestoides (Coleoptera: Lymexylidae), infesting a Quercus sp. (Davidson 1971). Some beetles in the latter genus are known to vector ambrosial yeasts (Batra and Francke-Grosmann 1961), but the role and biology of L. francke-grosmanniae in these galleries on oak remains unknown. If these beetle ecosystems in hardwoods are explored further, it seems reasonable to expect that additional species related to L. francke-grosmanniae could be discovered. These would most likely emerge as a species complex distinct from the L. olivaceum complex.

All species in the L. olivaceum complex, with the exception of L. francke-grosmanniae, share various characteristics. Apart from similar sexual and asexual morphology (as discussed in the introduction), these species are all associated with scolytine bark beetles infesting primarily species of pine (Pinus) and spruce (Picea). Only L. davidsonii has been reported from another conifer genus, namely Pseudotsuga (Douglas-fir). However, there is no evidence for strong host or beetle specificity among these fungi. The European spruce bark beetle, Ips typographus, for example, infests various species of spruce and pine, and L. cucullata, L. olivacea, and L. poloniae, have been isolated from this beetle or its galleries. Nothing is known regarding the pathogenicity of any of the species in the complex, but Griffin (1968) and Davidson (1958) showed that some species were responsible for the blue-stain of the timber.

In terms of the distribution of species in the L. olivaceum complex, our data suggest that most of these taxa are geographically restricted to the continents from which they have been recorded. Four species have been reported only from North America, namely L. davidsonii, L. olivaceapini, L. sagmatosporum, and L. vescum, while L. olivaceum, L. erubescens and four of the new species have been found only in Europe and western Russia. Two of the new species originate from China. Only L. cucullatum has been found in Europe and East Asia, specifically Japan.

The results of this study incorporating data for morphology, ecology, and phylogenetic inference based on DNA sequences for five loci have confirmed that the L. olivaceum complex is a well-defined species complex in Leptographium. Moreover, this integrative approach has been recently employed to resolve lower-level taxonomy in several other groups of fungi such as the Ophiocordycipitaceae (Araújo et al. 2015), Pyronemataceae (Sochorová et al. 2019), Laboulbeniaceae (Haelewaters et al. 2018), Geastraceae (Sousa et al. 2017), and Helvellaceae (Skrede et al. 2017). The combination of multiple properties as independent lines of evidence (e.g., morphology, DNA, substratum, and/or geography) is the way to move forward in fungal taxonomy in general.

Conclusions

In the present study, DNA sequences for five loci were amplified and used to reconstruct phylogenies for species in the L. olivaceum complex. Multilocus phylogenies distinguished clearly among the eight previously described species and also revealed six species: L. breviuscapum, L. conplurium, L. pseudoalbum, L. rhizoidum, L. sylvestris, and L. xiningense that are newly described. TEF-1α was recognized as the best candidate gene to distinguish all species in the complex. For several of the previously known species, problems relating to type specimens were identified, and to resolve these, seven new combinations, two epitypes and three lectotypes have been designated. Following the “one fungus one name” principles, this study provided a model solution to resolving interspecific relationships within the species complexes in the Ophiostomatales. More work should be done on other unresolved species complexes of Leptographium and other lineages in the Ophiostomatoid fungi in the future.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (31600025), Guangdong Province Natural Science Foundation of China (2017A030313138), as well as special funds for the cultivation of scientific and technological Innovation of college students in Guangdong Province (pdjh2019b0085). We acknowledge three reviewers (Prof. Yuichi Yamaoka, Prof. Georg Hausner, and Dr. Chase G. Mayers) for providing valuable comments that improved the paper. We are also grateful for support from members of the Tree Protection Cooperation Programme (TPCP) and the University of Pretoria, South Africa.

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Supplementary materials

Supplementary material 1 

The sequence alignment of combined four protein-coding gene regions

Mingliang Yin, Michael J. Wingfield, Xudong Zhou, Riikka Linnakoski, Z. Wilhelm de Beer

Data type: phylogenetic data.

Explanation note: The alignment was generated from MAFFT V7 Online, and it contained sequences of four protein coding genes (actin, beta-tubulin, calmodulin, and translation elongation factor 1 alpha) of all the isolates in the Leptographium olivacerum complex.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (100.65 kb)
Supplementary material 2 

The sequence alignment of ITS2-LSU gene region

Mingliang Yin, Michael J. Wingfield, Xudong Zhou, Riikka Linnakoski, Z. Wilhelm de Beer

Data type: phylogenetic data.

Explanation note: The alignment was generated from MAFFT V7 Online, and it contained sequences of Internal transcribed spacer 2 and large-subunit rRNA genes of all isolates used in this study.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (37.38 kb)
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