11urn:lsid:arphahub.com:pub:C004A564-9D6A-5F9F-B058-6A3815DFE9C3MycoKeysMC1314-40571314-4049Pensoft Publishers10.3897/mycokeys.60.3906939069Research ArticleOphiostomataceaeOphiostomatalesDNA barcodingPhylogenyTaxonomyAsiaFar EastTaxonomy and phylogeny of the Leptographiumolivaceum complex (Ophiostomatales, Ascomycota), including descriptions of six new species from China and EuropeYinMingliang12mingliang.yin@scau.edu.cnhttps://orcid.org/0000-0002-8811-3151WingfieldMichael J.2ZhouXudong3LinnakoskiRiikka24BeerZ. Wilhelm de2https://orcid.org/0000-0001-9758-8987Guangdong Key Laboratory for Innovative Development and Utilization of Forest Plant Germplasm, College of Forestry and Landscape Architecture, South China Agricultural University, Guangzhou, 510000, ChinaDepartment of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, Gauteng Province, South AfricaFuturaGene Biotechnology (Shanghai) Co. Ltd, Shanghai, 200233, ChinaNatural Resources Institute Finland (Luke), 00790 Helsinki, Finland
20192911201960931237E7DFCA0-FC45-5155-AE9E-1A325024250735660841408201922102019Mingliang Yin, Michael J. Wingfield, Xudong Zhou, Riikka Linnakoski, Z. Wilhelm de BeerThis 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.
The Leptographiumolivacea 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.
bark beetleLeptographiumintegrative taxonomynew speciesOphiostomatalesphylogenyNational Natural Science Foundation of China501100001809http://doi.org/10.13039/501100001809Citation
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
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 Leptographiumwageneri 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 Leptographiumsensu 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.
MethodsIsolates
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).
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
Leptographiumbreviscapum
38888 H
136507
China
Piceacrassifolia
Polygraphuspoligraphus
MN516697
MN517641
MN517672
MN517707
MN517742
38889 P
136508
China
Piceacrassifolia
Polygraphuspoligraphus
MN516698
MN517642
MN517673
MN517708
MN517743
38890
China
Piceacrassifolia
Polygraphuspoligraphus
MN516699
MN517643
MN517674
MN517709
MN517744
L.conplurium
23289 P
128834
Finland
Piceaabies
Dryocoetusautographus
MN516701
MN517644
JF279994
MN517710
MN517745
23295
Finland
Piceaabies
Dryocoetusautographus
MN516702
MN517645
JF279993
MN517711
JF280036
23315 H
128923
Finland
Piceaabies
Dryocoetusautographus
MN516700
MN517646
JF279989
MN517712
MN517746
23316
Finland
Piceaabies
Hylastesbrunneus
MN516703
MN517647
JF279990
MN517713
MN517747
L.cucullatum
1140=1141 H
218.83
Norway
Piceaabies
Ipstypographus
AJ538335
MN517619
JF280000
MN517685
MN517724
1871
Japan
Pinusjezoensis
Ipstypographus
MN516704
MN517620
JF280001
MN517686
MN517725
5022
Austria
Piceaabies
Ipstypographus
MN516705
MN517621
JF280002
MN517687
MN517726
23123
128299
Russia
Piceaabies
Ipstypographus
MN516706
MN517622
JF280003
MN517688
JF280042
23190
Russia
Pinussylvestris
Ipstypographus
MN516707
MN517623
JF280005
MN517689
JF280043
27983
Russia
Piceaabies
Dryocoetusautographus
MN516708
MN517624
MN517658
MN517690
MN517727
27984
Russia
Piceaabies
Dryocoetusautographus
MN516709
MN517625
MN517659
MN517691
MN517728
36623
Russia
Piceaabies
Ipstypographus
MN516710
MN517626
MN517660
MN517692
MN517729
L.davidsonii
790 H
Canada
Pseudotsugamenziesii
–
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
Pinussylvestris
–
MN516714
MN517656
MN517683
MN517722
MN517756
L.francke-grosmanniae
445 H
356.77
Germany
Quercus sp.
Hylecoetusdermestoides
MN516715
MN517618
MN517657
MN517684
MN517723
L.olivaceum
23348
128836
Finland
Piceaabies
Ipstypographus
MN516717
MN517630
MN517664
MN517696
JF280049
23350
128837
Finland
Piceaabies
Ipstypographus
MN516718
MN517631
MN517665
MN517697
JF280050
28090
Russia
Pinussylvestris
Ipstypographus
MN516719
MN517632
MN517666
MN517698
MN517733
31059 H
138.51
Sweden
Pinussylvestris
–
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
Pinussylvestris
Tomicuspiniperda
MN516723
MN517655
MN517682
MN517721
MN517755
L.rhizoidum
22809 H
136512
Spain
Pinusradiata
Hylastesater
MN516724
MN517648
MN517675
MN517714
MN517748
22810 P
136513
Spain
Pinusradiata
Hylastesattenuatus
MN516725
MN517649
MN517676
MN517715
MN517749
22811
Spain
Pinusradiata
Ipssexdentatus
MN516726
MN517650
MN517677
MN517716
MN517750
22812
Spain
Pinusradiata
Hylurgopspalliatus
MN516727
MN517651
MN517678
MN517717
MN517751
L.sagmatosporum
34135 E
113452
Canada
Pinusstrobus
–
MN516728
MN517637
MN517669
MN517703
MN517738
L.sylvestris
23300 P
128833
Finland
Piceaabies
Ipstypographus
MN516729
MN517639
JF279996
MN517705
MN517740
34140 T
136511
Poland
Pinussylvestris
–
MN516730
MN517640
MN517671
MN517706
MN517741
L.vescum
34186 H
800.73
USA
Piceaengelmannii
Ipspilifrons, Dendroctonusengelmanni
MN516731
MN517638
MN517670
MN517704
MN517739
L.xiningense
38891 H
136509
China
Piceacrassifolia
Polygraphuspoligraphus
MN516732
MN517652
MN517679
MN517718
MN517752
39237 P
136510
China
Piceacrassifolia
Polygraphuspoligraphus
MN516733
MN517653
MN517680
MN517719
MN517753
39238
China
Piceacrassifolia
Polygraphuspoligraphus
MN516734
MN517654
MN517681
MN517720
MN517754
1Bold type = new species in the present study. 2CMW = 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 3ITS2 = 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 Fragosphaeriapurpurea 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.
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 (Pinuspinaster) 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).
ResultsPhylogenetic 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. Leptographiumfrancke-grosmanniae grouped peripheral to other species in the complex, but remained part of a strongly supported lineage including all the species under consideration.
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.
https://binary.pensoft.net/fig/360780
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.
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.
https://binary.pensoft.net/fig/360781
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.
FungiOphiostomatalesOphiostomataceae06018524-9DDB-5459-9CF5-1A1BFB961A6BLeptographiumbreviuscapum823576M.L. Yin, Z.W. de Beer & M.J. Wingf.sp. nov.Fig. 3Etymology.
The epithet (brevius-, short, and -scapum, branch) refers to very short conidiophores.
Type.
CHINA, Qinghai province, from Piceacrassifolia infested with Polygraphuspoligraphus, Aug. 2010, M.L. Yin & X.D. Zhou, (PREM 60914 holotype, ex-holotype cultures CBS 136507 = CMW 38888); Qinghai province, from Piceacrassifolia 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.
Leptographiumbreviuscapum sp. nov. (CMW 38888) a fourteen-days old culture on OA with black background b synnematous asexual state on wood tissue on WAc–d conidiophore e conidiogenous cells f conidia. Scale bars: 100 μm (b), 25 μm (c), 25 μm (d), 10 μm (e), 5 μm (f).
https://binary.pensoft.net/fig/360782Host tree.
Piceacrassifolia.
Insect vector.
Polygraphuspoligraphus.
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 Piceacrassifolia infested with Polygraphuspoligraphus, Aug. 2010, M.L. Yin & X.D. Zhou, (culture: CMW 38890). Yunnan province, from Pinusyunnanensis infested with Tomicusyunnanense, Sep. 2017, M.L. Yin, (culture: SCAU-475). Yunnan province, from Pinusyunnanensis infested with Tomicusyunnanense, Sep. 2017, M.L. Yin, (culture: SCAU-478).
FungiOphiostomatalesOphiostomataceae0D08C846-A29F-5447-AFD6-7AA0D062CE28Leptographiumconplurium823572M.L. Yin, Z.W. de Beer & M.J. Wingf.sp. nov.Fig. 4Etymology.
The epithet refers to synnemata produced abundantly in culture.
Type.
FINLAND, Ilomantsi, from Piceaabies infested with Dryocoetesautographus, 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.
Leptographiumconplurium sp. nov. (CMW 23315). a fourteen-days old culture on OA with black background; b. synnematous asexual state on wood tissue on WAc 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).
https://binary.pensoft.net/fig/360783Host tree.
Piceaabies.
Insect vectors.
Dryocoetesautographus, Hylastesbrunneus.
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 Piceaabies infested with Dryocoetesautographus, Aug. 2005, Z.W. de Beer, (culture: CMW 23295); Ilomantsi, from P.abies infested with Hylastesbrunneus, Aug. 2005, Z.W. de Beer, (culture: CMW 23316).
FungiOphiostomatalesOphiostomataceae63322A10-8802-5AD9-BFE8-0C385FAC3096Leptographiumcucullatum831546(H. Solheim) M.L. Yin, Z.W. de Beer & M.J. Wingf.comb. nov.≡ Ophiostomacucullatum H. Solheim, Nord. J. Bot. 6: 202 (1986). (Basionym) ≡ Grosmanniacucullata (H. Solheim) Zipfel, Z.W. de Beer & M.J. Wingf., Zipfel et al. (2006) Stud. Mycol. 55: 90. Type.
NORWAY, Vestfold, Lardal, from Ipstypographus caught when leaving a log of Piceaabies, 20 Aug 1981, H. Solheim, (CBS H-15306 and CBS H-3560-holotype, ex-holotype cultures: CMW 1140 = CBS 218.83 = 81-83/16).
Harrington et al. (2001) suggested that Phialographiumerubescens 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 Piceaabies, July 1997, T. Kirisits, CMW 5022; JAPAN, Hokkaido, Furano, from an adult of Ipstypographusjaponicus in Piceajezoensis, 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 Pinussylvestris, R. Linnakoski, CMW 23190; RUSSIA, Kivennapa, Lintula, from Dryocoetusautographus in P.abies, Oct 2007, R. Linnakoski, CMW 27983, CMW 27984; RUSSIA, Karelia, from I.typographus in P.abies, H. Roininen, CMW 36623.
FungiOphiostomatalesOphiostomataceae6DDD1B84-0353-55BC-AFBD-16B797429197Leptographiumdavidsonii831547(Olchow. & J. Reid) M.L. Yin, Z.W. de Beer & M.J. Wingf.comb. nov.≡ Ceratocystisdavidsonii (Olchow. & J. Reid), Can. J. Bot. 52: 1698 (1974). (Basionym) ≡ Ophiostomadavidsonii (Olchow. & J. Reid) H. Solheim, Nord. J. Bot. 6: 203 (1986). ≡ Grosmanniadavidsonii (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 Pseudotsugamenziesii, 1971, J. Reid, (WIN (M) 71-30-holotype, ex-holotype cultures: CMW 790 = IMI 176524 = JCM 7867).
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 Dryocoeteshectographus on Abiesveitchii 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).
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 Pinussylvestris, 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).
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 Graphiumerubescens (as Phialographiumerubescens) 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.
GERMANY, Reinbeck near Hamburg, from Quercus sp. associated with Hylecoetusdermestoides, 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.
Hylecoetusdermestoides.
Distribution.
Germany.
Notes.
Leptographiumfrancke-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). Leptographiumfrancke-grosmanniae is also unusual in the L.olivaceum complex in having an angiosperm host.
Leptographiumfrancke-grosmanniae was originally described as Ceratocystisfrancke-grosmanniae from larval galleries of Hylecoetusdermestoides 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.
SWEDEN, Hällnäs, Västerbotten, from the galleries of Acanthocinusaedilis 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 Pinussylvestris, 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).
This species was first described invalidly (no Latin diagnosis) from Pinussylvestris infested by a longhorn beetle Acanthocinusaedilis 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 Piceaabies and Pinussylvestris infested by Ipstypographus and Dryocoetesautographus 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 Ipstypographus in Piceaabies, July 2005, Z.W. de Beer, (cultures: CMW 23348 = CBS 128836, CMW 23350 = CBS 128837). RUSSIA, Uuksujärvi, from I.typographus in Pinussylvestris, Oct 2007, R. Linnakoski, (culture CMW 28090). SWEDEN, Oct 1954, A. Mathiesen-Käärik, (cultures: CMW 31060 = CBS 152.54).
FungiOphiostomatalesOphiostomataceaeAC704DF1-9357-5B49-8AAA-DAED89C4592DLeptographiumolivaceapini831549(R.W. Davidson) M.L. Yin, Z.W. de Beer & M.J. Wingf.comb. nov.≡ Ceratocystisolivaceapini R.W. Davidson, Mycologia 63: 7 (1971). (Basionym) ≡ Ophiostomaolivaceapini (R.W. Davidson) K.A. Seifert & G. Okada, In Okada et al., Can. J. Bot. 76: 1504 (1998). ≡ Grosmanniaolivaceapini (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 Pinusponderosa 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 Pinusponderosa 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, (epitypePREM 61051, designated here, ex-epitype cultures CBS 504.86 = CMW 116 = COLO 479, MBT 379458).
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, Pinusresinosa, Nov 1986, M.J. Wingfield, (cultures: CBS 503.86 = CMW 63).
FungiOphiostomatalesOphiostomataceae4ECD53D1-B9F3-5404-A478-7EABEC9BF2FBLeptographiumpseudoalbum823571M.L. Yin, Z.W. de Beer and M.J. Wingf.sp. nov.Fig. 5Etymology.
The epithet refers to the previous, incorrect identification of the ex-holotype isolate of this species as Graphiumalbum.
Type.
SWEDEN, from Pinussylvestris infested by Tomicuspiniperda, 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.
Leptographiumpseudoalbum sp. nov. (CBS 276.54) a fourteen-days old culture on OA with black background; b. synnematous asexual state on wood tissue on WAc–d conidiophore e conidiogenous cells f conidia. Scale bars: 200 μm (b), 25 μm (c), 25 μm (d), 10 μm (e), 5 μm (f).
https://binary.pensoft.net/fig/360784Host.
Pinussylvestris.
Insect vector.
Tomicuspiniperda.
Distribution.
Sweden.
Notes.
This species was initially identified as Graphiumalbum (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.
FungiOphiostomatalesOphiostomataceaeD9A34037-97F6-5AD7-87CE-CAA88E1293B7Leptographiumrhizoidum823575M.L. Yin, Z.W. de Beer and M.J. Wingf.sp. nov.Fig. 6Etymology.
The epithet refers to the rhizoid-like structures at the synnematal bases.
Type.
SPAIN, Morga, from Pinusradiata infested by Hylastesater, July. 2004, P. Romon & X.D. Zhou, (PREM 60922-holotype, ex-holotype cultures: CBS 136512 = CMW 22809); Morga, from Pinusradiata infested by Hylastesattenuatus, 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.
Leptographiumrhizoidum sp. nov. (CMW 22809). a fourteen-days old culture on OA with black background b synnematous asexual state on wood tissue on WAc 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).
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 Pinusradiata infested by Ipssexdentatus, July. 2004, P. Romon & X.D. Zhou, (culture: CMW 22811); Morga, from P.radiata infested by Hylurgopspalliatus, July. 2004, P. Romon & X.D. Zhou, (culture: CMW 22812).
FungiOphiostomatalesOphiostomataceae88DA1577-F14D-5B3E-86B5-EDA29C32EDA5Leptographiumsagmatosporum831550(E.F. Wright & Cain) M.L. Yin, Z.W. de Beer & M.J. Wingf.comb. nov.≡ Ceratocystissagmatospora E.F. Wright & Cain, Can. J. Bot. 39: 1226 (1961). (Basionym). ≡ Phialographiumsagmatosporae H.P. Upadhyay and W.B. Kendr., Mycologia 66: 183 (1974). ≡ Ophiostomasagmatosporum (E.F. Wright & Cain) H. Solheim, Nord. J. Bot. 6: 203 (1986). ≡ Graphiumsagmatosporae (H.P. Upadhyay & W.B. Kendr.) M.J. Wingf. & W.B. Kendr., Mycol. Res. 95: 1332 (1991). ≡ Pesotumsagmatosporum (H.P. Upadhyay & W.B. Kendr.) G. Okada & K.A. Seifert, in Okada et al., Can. J. Bot. 76: 1504 (1998). ≡ Grosmanniasagmatospora (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 Pinusresinosa, 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 Pinusstrobus, Sept. 2000, K. Jacobs, (epitypePREM 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.
Pinusstrobus, Piceamariana.
Insect vectors.
unknown bark beetle species.
Distribution.
Canada.
Notes.
This species was originally described from bark beetle galleries and freshly cut surfaces of Piceamariana, Pinusresinosa and Pinusstrobus 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 Pinusresinosa, Nov. 8 1958, E.F. Wright & R.F. Cain, TRTC 34600; NW. of Nobleton, York Co., from Pinusstrobus, July 1 1957, E.F. Wright & R.F. Cain, TRTC 33034; Twp. West of 11 H, Challener Lake, Sudbury Dist., from Pinusstrobus, June 20 1960, E.F. Wright & R.F. Cain, TRTC 36245, 36251, 36255, 36264, 36265;Twp. 5F, Aubinadong R., Algoma Dist, from Pinusstrobus, June 17 1960, E.F. Wright & R.F. Cain, TRTC 36246; Twp. West of 11 H, Challener Lake, Sudbury Dist., from Piceamariana, June 20 1960, E.F. Wright & R.F. Cain, TRTC 36263.
FungiOphiostomatalesOphiostomataceaeB4B99C40-4783-5175-B8C8-81DD0CF31211Leptographiumsylvestris823574M.L. Yin, Z.W. de Beer and M.J. Wingf.sp. nov.Fig. 7Etymology.
The epithet refers to the host species where the holotype was collected.
Type.
POLAND, Chrosnica, from Pinussylvestris, Jan. 2008, R. Jankowiak, (PREM 60920-holotype, ex-holotype cultures: CBS 136511 = CMW 34140). FINLAND, Jouhteninen, from Piceaabies infested with Ipstypographus, 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.
Leptographiumsylvestris sp. nov. (CMW 34140) a fourteen-days old culture on OA with black background b synnematous asexual state on wood tissue on WAc conidiophore d conidiogenous apparatus e conidiogenous cells f conidia g–h the sexual state on wood tissue on WAi 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).
https://binary.pensoft.net/fig/360787Host trees.
Pinussylvestris, Piceaabies.
Insect vector.
Ipstypographus.
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.
USA, Colorado, Fort Collins, from Ipspilifrons and Dendroctonusengelmanni in Piceaengelmannii, 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.
Piceaengelmannii.
Insect vectors.
Ipspilifrons, Dendroctonusengelmanni.
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.
FungiOphiostomatalesOphiostomataceae6A650550-E056-54A7-ADAA-861D04522970Leptographiumxiningense823573M.L. Yin, Z.W. de Beer and M.J. Wingf.sp. nov.Fig. 8Etymology.
The epithet refers to the locality where the species was first collected.
Type.
CHINA, Qinghai Province, from Piceacrassifolia infested by Polygraphuspoligraphus, Aug. 2010, M.L. Yin & X.D. Zhou, (PREM 60916-holotype, ex-holotype cultures CBS 136509 = CMW 38891); Qinghai Province, from Piceacrassifolia infested by Polygraphuspoligraphus, 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.
Leptographiumxiningense sp. nov. (CMW 38891) a fourteen-days old culture on OA with black background b synnematous asexual state on wood tissue on WAc 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).
https://binary.pensoft.net/fig/360788Host tree.
Piceacrassifolia.
Insect vector.
Polygraphuspoligraphus.
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 Piceacrassifolia infested by Polygraphuspoligraphus, Aug. 2010, M.L. Yin & X.D. Zhou, (culture: CMW 39238). Chongqing, from Pinusarmandii infested by Dendroctonusarmandi, Nov. 2018, M.L. Yin, (culture: SCAU-530). Chongqing, from Pinusarmandii infested by Dendroctonusarmandi, 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, Hylecoetusdermestoides (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, Ipstypographus, 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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The sequence alignment of combined four protein-coding gene regions
phylogenetic data.
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.
https://binary.pensoft.net/file/360789This 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.Mingliang Yin, Michael J. Wingfield, Xudong Zhou, Riikka Linnakoski, Z. Wilhelm de Beer10.3897/mycokeys.60.39069.suppl23566104CE1072CD-8A7B-5DDD-9940-C4D840991B60
The sequence alignment of ITS2-LSU gene region
phylogenetic data.
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.
https://binary.pensoft.net/file/360790This 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.Mingliang Yin, Michael J. Wingfield, Xudong Zhou, Riikka Linnakoski, Z. Wilhelm de Beer