MycoKeys 33: | 8 1-208 (202 | ) r-reviewed open-access journa

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Ophiostomatoid species associated with pine trees (Pinus spp.) infested by Cryphalus piceae from eastern China, including five new species

Runlei Chang', Xiuyu Zhang', Hongli Si', Guoyan Zhao', Xiaowen Yuan’, Tengteng Liu', Tanay Bose’, Meixue Dai!

I College of Life Sciences, Shandong Normal University, Jinan 250014, China 2. Kunyushan Forest Farm, Yan- tai 264112, China 3 Forestry and Agricultural Biotechnology Institute (FABI), Department of Biochemistry, Genetics & Microbiology, University of Pretoria, Pretoria 0002, South Africa

Corresponding author: Meixue Dai (daimeixue@sdnu.edu.cn)

Academic editor: Nattawut Boonyuen | Received 1 July 2021 | Accepted 20 September 2021 | Published 13 October 2021

Citation: Chang R, Zhang X, Si H, Zhao G, Yuan X, Liu T, Bose T, Dai M (2021) Ophiostomatoid species associated with pine trees (Pinus spp.) infested by Cryphalus piceae from eastern China, including five new species. MycoKeys 83: 181-208. https://doi.org/10.3897/mycokeys.83.70925

Abstract

Cryphalus piceae attacks various economically important conifers. Similar to other bark beetles, Cx piceae plays a role as a vector for an assortment of fungi and nematodes. Previously, several ophiostomatoid fungi were isolated from C7. piceae in Poland and Japan. In the present study, we explored the diversity of ophiostomatoid fungi associated with C7. piceae infesting pines in the Shandong Province of China. We isolated ophiostomatoid fungi from both galleries and beetles collected from our study sites. These fungal isolates were identified using both molecular and morphological data. In this study, we recovered 175 iso- lates of ophiostomatoid fungi representing seven species. Ophiostoma ips was the most frequently isolated species. Molecular and morphological data indicated that five ophiostomatoid fungal species recovered were previously undescribed. Thus, we proposed these five novel species as Ceratocystiopsis yantaiensis, C. weihaiensis, Graphilbum translucens, Gr. niveum, and Sporothrix villosa. These new ophiostomatoid fungi add to the increasing number of fungi known from China, and this evidence suggests that numerous

novel taxa are awaiting discovery in other forests of China.

Keywords Ceratocystiopsis, fungal symbionts, Graphilbum, nematode vector, Ophiostoma, Sporothrix

Copyright Runlei Chang et al. 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.

182 Runlei Chang et al. / MycoKeys 83: 181—208 (2021)

Introduction

Wingfield et al. (1993) coined the name “ophiostomatoid fungi” referring to a poly- phyletic group of fungi that included several species from the orders Microascales and Ophiostomatales. These fungi are distinguished by spores generated in sticky droplets that aid in dispersion by arthropods (De Beer et al. 2013). The order Microascales includes three families, including Ceratocystidaceae (11 genera), Gondwanamycetaceae (2 genera), and Graphiaceae (1 genus) (De Beer et al. 2013). The Ophiostomatales was divided into two families: Ophiostomataceae (11 genera) and Kathistaceae (3 genera) (Hyde et al. 2020). Initially, De Beer and Wingfield (2013) identified 18 species com- plexes within the order Ophiostomatales. Later, the ‘S. schenckii — O. stenoceras’ species complex was elevated to genus level as Sporothrix (De Beer et al. 2016). Subsequently, this genus was divided into six species complexes (De Beer et al. 2016). Following this, Linnakoski et al. (2016a), Yin et al. (2016), and Jankowiak et al. (2017b) identified the O. clavatum, O. piceae and G. grandifoliae species complexes, respectively. Currently, the order Ophiostomatales thus encompasses at least 26 species complexes (De Beer and Wingfield 2013; De Beer et al. 2016; Linnakoski et al. 2016a; Yin et al. 2016; Jankowiak et al. 2017b).

Ophiostomatoid fungi often form a symbiotic association with bark and ambrosia beetles who assist in the dispersal of their inocula (Klepzig and Six 2004). For example, Ceratocystiopsis ranaculosus colonizes the mycangium of Dendroctonus frontalis whereas Ophiostoma minus is carried phoretically on the exoskeleton (Hofstetter et al. 2015). In addition, an ophiostomatoid fungus can symbiotically associate with multiple beetle species. Recently, six ophiostomatoid fungi were isolated from Monochamus alternatus in China (Zhao et al. 2014; Wang et al. 2018). Among them, Ophiostoma ips was previous- ly isolated from Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle and M. alternatus Hope from North America and Korea, respectively (Wingfield 1987; Suh et al. 2013).

Beetle-associated ophiostomatoid fungi play pivotal roles in the ecosystem. As ex- emplified by Endoconidiophora polonica and Sporothrix sp. 1., these fungi can provide beetles with nourishment, help them overcome plant defenses, and increase their vi- tality (Hammerbacher et al. 2013; Zhao et al. 2013; Wadke et al. 2016). Endoconidi- ophora polonica uses plant defensive compounds such as stilbenes and flavonoids as a carbon source, whilst Sporothrix sp. 1. enhances the development and survival rate of arthropods such as MV. alternatus (Zhao et al. 2013). This evidence confirms that ophi- ostomatoid fungi substantially influence the devastation caused by these arthropods in forestry contexts globally.

In Europe and Asia, Cryphalus piceae infests various species of Abies, Pinus, Pi- cea, and Larix (Jankowiak and Kolarik 2010). This bark beetle predominantly affects stressed trees (Michalski and Mazur 1999), but can also attack healthy ones (Justesen et al. 2020). Previously, several fungal species were isolated from C7. piceae infesting Abies alba and A. veitchii. This data included an assortment of ophiostomatoid fungi from the genera Graphilbum, Grosmannia, Leptographium, Ophiostoma, and Sporothrix from Poland (JJankowiak and Kolarik 2010; Jankowiak et al. 2017a) and Japan (Oh-

Ophiostomatoid fungi associated with Cryphalus piceae in China 183

taka et al. 2002a; Ohtaka et al. 2002b), and hypocrealean species from the genus Geo- smithia from Poland (Jankowiak and Bilanski 2018).

In China, knowledge regarding the diversity of ophiostomatoid fungi associated with C7. piceae is currently limited. Between 2019 and 2020, we thus conducted sur- veys of numerous Pinus stands in China's Shandong province. During these surveys, we collected samples of wood and bark from afflicted trees that had beetle galleries. From these samples, 175 isolates of ophiostomatoid fungi were isolated. Analyses of molecu- lar and morphological data revealed that our isolates belonged to seven different spe- cies of ophiostomatoid fungi. Among these, phylogenetic and morphological analyses confirmed that five of these taxa from China were previously undescribed. Here we described these species as Ceratocystiopsis yantaiensis sp. nov., C. weihaiensis sp. nov., Graphilbum translucens sp. nov., Gr. niveum sp. nov., and Sporothrix villosa sp. nov.

Materials and methods

Collection of beetles and isolation of fungi

From September 2019 to August 2020, multiple surveys were conducted in several Pinus thunbergii stands located near Weihai (37°30'07"N, 121°07'24"E) and Yantai (37°15'38"N, 121°44'39"E), and Pinus densiflora located near Qingdao (36°15'26"N, 121°38'07"E), Shandong Province of China. All these Pinus thunbergii and Pinus den- siflora stands were infested by Cr. Piceae along with Bursaphelenchus xylophilus and Monochamus alternatus. Samples of wood and bark with beetle galleries were collected from affected trees. In the laboratory, adult beetles from these galleries were individu- ally collected in 2 ml sterile collection tubes inside a laminar flow cabinet. Both galler- ies and beetles were stored at 4 ‘C until the isolation of fungi.

Beetles were identified using both morphological and molecular data. In the case of the latter, cytochrome oxidase subunit I (COD) was used as the marker gene re- gion. Sequences of bark beetle were identified using the “animal identification [COI]” database available through BOLDSYSTEMS (https://v3.boldsystems.org/). Sequence similarity searches confirmed the identity of all bark beetles as Cx piceae. Hence, two representative sequences of the bark beetle were submitted to GenBank under the ac- cession numbers MZ778788 and MZ778789.

In total, 32 adult beetles and 89 galleries were used for the isolation of ophios- tomatoid fungi. Fungal isolation was done using the method suggested by Chang et al. (2019). Fungal mycelia and/or spore masses from C7. piceae galleries were transferred onto 2% malt extract agar (MEA, Qingdao Hope Bio-technology, Qingdao, China) medium amended 0.05% streptomycin (Sangon Biotech, Qingdao, China). In cases of no mycelia and/or spore masses, galleries were incubated in moist chambers at 25 °C in darkness for 4—6 weeks. Post incubation, conidia with spore masses emerging from the conidiophores were transferred onto MEA amended with 0.05% streptomycin. To isolate ophiostomatoid fungi from the beetles, adult C7 piceae was crushed on a sterile

184 Runlei Chang et al. / MycoKeys 83: 181-208 (2021)

Table |. Isolates of ophiostomatoid fungi isolated from Cryphalus piceae in this study.

Isolate |CGMCC] Tree host | Location CAL Ceratocystiopsis | SNM582 Pinus Gallery |} MW989410 | MZ819923 | MZ019522 | MZ853079 -

yantaiensis sp. thunbergii nov. SNM650° | 3.20247 | P thunbergii Gallyer | MW/989411 | MZ819924 | MZ019523 | MZ853080 - Ceratocystiopsis P. thunbergii | Weihai | Gallery | MW989412 | MZ819925 | MZ019524| MZ853081 -

weihaiensis sp. | SNM649" | 3.20246 | P thunbergii | Weihai | Gallery | MW989413 | MZ819926 | MZ019525 | MZ853082 =

Graphilbum P thunbergii | Weihai_| Gallery | MW989414| — _ |MZ019526|MZ019544|MZ781969 translucens sp. P densiflora | Qingdao | Gallery | MW989415| — __ |MZ019527|MZ019545|MZ781970

~ 3.20263 | P thunbergii| Weihai | Gallery |} MW989416 MZ019528 | MZ019546 | MZ781971 Graphilbum P densiflora | Qingdao | Gallery | MW989417 MZ019529 | MZ019547 | MZ418998 niveum sp. SNM145"| 3.50268 | P thunbergii Beetle | MW989418 MZ019530 | MZ019548 | MZ418997

nov. Graphium SNM159 P. thunbergii | Weihai | Gallery | MW989419 MZ019549 -

pseudormiticum

Opp Pande | Wei | Gallery [RVSRDDO| —_— _[Mamiossi| _— | _— Pe dnbegi | Web| Gallery [woa94a1| —— | zoi9ss2[_—- | _—

P chunbergii | Weihai_| Beetle |MW989425| — — _ |MZ019536|MZ853075 | MZ019540 P thunbergii | Weihai | Beetle |MW989426| — | MZ019537|MZ853076]MZ019541

Sporothrix

villosa sp. nov.

S S S 5S S S

surface using a pair of forceps, thereafter, this crushed beetle was placed on the surface of MEA amended 0.05% streptomycin. To purify the fungal isolates, hyphal tips from fungal colonies were transferred onto fresh MEA plates.

All fungal isolates were submitted to the microbial culture collection of Shan- dong Normal University, Jinan, Shandong, China (SNM; for accession numbers see Table 1). Ex-holotypes cultures of ophiostomatoid fungi described in this study were deposited in the China General Microbiological Culture Collection Center (CGM- CC; hetp://www.cgmcc.net/english/catalogue.html), Beijing, China. Holotype speci- mens (dry cultures) were deposited in the Herbarium Mycologicum, Academiae Sini-

cae (HMAS), Beijing, China.

DNA extraction, PCR amplification and sequencing

All fungal isolates obtained in this study were initially grouped based on colony mor- phology. For preliminary identification, at least two representative isolates from each group were identified using molecular techniques. For the novel species described in the present study, all isolates were sequenced to confirm their identity.

The PrepMan ultra sample preparation reagent (Applied Biosystems, Foster City, CA) was used for extracting the total genomic DNA from five-day-old cultures, fol- lowing the manufacturer's protocols. The complete ITS region, and partial large subu- nit (LSU) of the nuclear ribosomal RNA (rRNA) gene, and partial 8-tubulin (BT), elongation factor 1-« (EF), and calmodulin (CAL) genes were amplified using primers ITSIF/ITS4 (White et al. 1990; Gardes and Bruns 1993), LROR/LR5 (Vilgalys and Hester 1990), Bt2a (or T'10)/Bt2b (Glass and Donaldson 1995), EF2F/EF2R (Jacobs

Ophiostomatoid fungi associated with Cryphalus piceae in China 185

et al. 2004; Marincowitz et al. 2015), and CL2F/CL2R (Duong et al. 2012), respec- tively.

Each 25 pl PCR reaction included 12.5 pl 2 x Taq Master Mix (buffer, dNTPs, and Taq; Vazyme Biotech Co., Ltd, China), 0.5 pl each of forward and reverse prim- ers, 10.5 pl PCR grade water, and 1 ul of DNA template. PCR amplifications were conducted with an initial denaturation at 95 °C for 3 min, followed by 30 cycles of 95 °C for 60 sec; annealing temperature was 55 °C for 60 sec for all primers; 72 °C for 1 min; and final elongation at 72 °C for 10 min.

All PCR products were sequenced by Sangon Biotech, Qingdao, Shandong Prov- ince, China. The sequences were assembled using Geneious v. 7.1.4 (Biomatters, Auck- land, New Zealand). The BLAST algorithm (Altschul et al. 1990) available through the NCBI GenBank was used for the preliminary identification of the taxa. All se- quences were submitted to GenBank and the accession numbers are listed in Table 1.

Phylogenetic analyses

For phylogenetic analyses, separate datasets were prepared for all four gene regions (ITS, BT, EF and CAL). Each of these datasets included sequences generated in this study, and those that were retrieved from the GenBank (including the ex-type se- quences, Suppl. material 3: Table S1). We recovered a high number of isolates rep- resenting the same species from O. ips (141 isolates) and S. gossypina species complex (24 isolates). Therefore, datasets for these two species complexes included sequences from four representative isolates. The gene areas that are available in public databases substantially vary amongst genera and species complexes of ophiostomatoid fungi. As a result, we chose gene regions for our study based on previous research. ‘These are as follows: ITS, BT, EF and CAT for Graphilbum (Jankowiak et al. 2020), ITS and BT for O. ips species complex (Wang et al. 2020), ITS, LSU and BT for Ceratocystiopsis (Nel et al. 2021), ITS, BT and CAL for Sporothrix (De Beer et al. 2016; Wang et al. 2018), and ITS and EF for Graphium (Chang et al. 2019). The datasets were aligned using MAFFT v. 7 (Katoh and Standley 2013). If needed, alignments were manually edited using MEGA v. 6.06 (Tamura et al. 2013). All aligned sequence datasets were deposited to TreeBase (Acc. No. 28127).

Programs used for maximum likelihood (ML) and Bayesian inference (BI) analyses were accessed through the CIPRES Science Gateway v. 3.3 (Miller et al. 2010). For all datasets, jModel Test v. 2.1.6 (Darriba et al. 2012) was used for selecting appropriate substitution models. Maximum likelihood analyses were done through RaxML v. 8.2.4 (Stamatakis 2014) using the GTR substitution model and 1000 bootstrap replicates. Bayesian inference analyses were done using MrBayes v. 3.2.6 (Ronquist et al. 2012). Four MCMC chains were run from a random starting tree for five million genera- tions and trees were sampled every 100" generation. One-fourth of the sampled trees were discarded as burn-in and the remaining trees were used for constructing majority rule consensus trees. MEGA-X was used for conducting maximum parsimony (MP) analyses with 1000 bootstrap replicates (Kumar et al. 2018) where gaps were treated as a fifth character.

186 Runlei Chang et al. / MycoKeys 83: 181—208 (2021)

Growth and morphological studies

For each new fungal species, an ex-type along with another isolate identified through phylogenetic analyses were selected for growth study. Isolates were initially sub-cultured on 2% MEA and incubated for seven days at 25 °C in darkness. ‘Thereafter, 5 mm agar plugs were placed at the center of 90 mm Petri dishes and three replicate plates per isolate were incubated at 5, 10, 15, 20, 25, 30 and 35 °C (+ 0.5 °C) in darkness. The colony diameter of each isolate was measured at an interval of two days up to the tenth day.

Microscopic structures of the ophiostomatoid fungi were measured and photo- graphed using a Zeiss Axio Imager Z2 (CarlZeiss, Germany). Fifty measurements for each taxonomically informative structure were made, such as conidiophore and conid- ia. Ihe measurements are presented in the format (minimum—) mean minus standard deviation-mean plus standard deviation (maximum).

Results

Collection of beetles and isolation of fungi

In the present study, 175 isolates of ophiostomatoid fungi were recovered. Among these, 148 were isolated from galleries, whereas 28 were from beetles. Based on the collection sites, 16, 63, and 96 isolates were recovered from Yantai, Qingdao, and Weihai, respectively.

Phylogenetic analyses

Preliminary identification of the ophiostomatoid fungi recovered in this study showed that the isolates resided in the genera Ceratocystiopsis (4 isolates), Graphilbum (5 iso- lates), Graphium (1 isolate), Ophiostoma (141 isolates), and Sporothrix (24 isolates).

Species residing in the genus Ceratocystiopsis were analyzed using ITS, LSU, and BT gene regions. In the phylogenies of Ceratocystiopsis, four isolates of Ceratocystiopsis clustered into two distinct monophyletic clades (Figs 1 and 2). Taxon 1 (two isolates) and Taxon 2 (two isolates) were found to be sisters to C. manitobensis and C. minuta, respectively (Figs 1 and 2).

Species residing in the genus Graphilbum were analyzed using ITS, BT, CAL, and EF gene regions. The taxon sampling differed substantially amongst the gene regions due to the lack of sequences. In the phylogenetic analyses, our five isolates of Graphilbum clustered into two distinct clades (Figs 3-5). The three isolates of Taxon 3 nested within clades that included Gr. acuminatum, Gr. anningense, and Gr. puerense (Figs 3-5). In the ITS, CAL, and EF trees, the two isolates of Taxon 4 were found to be closely related to Gr. crescericum (Figs 3-5). In contrast, Taxon 4 emerged as the sister species to Gr. kesiyae in the BT tree (Fig. 4). This is due to the lack of BT gene sequences for Gx. crescericum.

The identity of the isolate residing in Graphium was confirmed using ITS and EF gene regions. In the phylogenies, the single isolate of Taxon 5 emerged as a previously described species, G. pseudormiticum (Suppl. material 1).

Ophiostomatoid fungi associated with Cryphalus piceae in China 187

100/100 essen ITS 100/99

C. manitobensis UM214 C. manitobensis UM237 93/97] C. manitobensis 3PG2P-Om C. manitobensis 3YT3P-Om 84/-| 99/96, © ‘Tanaculosa CBS 216.88 C. ranaculosa 94-P320 98/97), C. ranaculosa C1245 C. brevicomis UM1452 b C. collifera CBS 126.89 98/1100) Ceratocystiopsis sp. WY13TX1-3 96/994 Ceratocystiopsis sp. WY21TX2-2 Ceratocystiopsis sp. WY21TX1-2

76/-

100/100 fl 87/94 i ow ] MIC. minuta

2 C. minuta UM1535

C. minima UM235

C. minima UM85

99/100 Ceratocystiopsis sp. YCC330

100/99 Ceratocystiopsis sp. YCC329

100/100 [C. minima UM844

94/99 C. minuta-bicolor UM480

C. minuta-bicolor CBS 635.66 _ C. pallidobrunnea WIN M 51

C. synnemata KFL17718 100/100'C. synnemata KFL16918

100/100

100/100 C. lunata CMW55898 C. lunata CMW55897 T 100/100 C. rollhanseniana UM110

C. rollhanseniana UM113 Ophiostoma bacillisporum CBS 771.71 T

0.05 100/99) &

a 92/84 | C. yantaiensis SNM582 — C. manitobensis UM214

100/100 Lc. manitobensis UM237 78188 J _La{C. manitobensis 3PG2P-Om 400/100 + C. manitobensis 3YT3P-Om

100/100 |C. minuta-bicolor UM480

C. minuta-bicolor UM844

C. minuta-bicolor CBS635.66

100/100 76181 S h i bla 3

-/91|_ LiC. minuta CBS116796

fem C. minuta UM1535 Ceratocystiopsis sp. YCC329

100/100

100/100 100/100

Ceratocystiopsis sp. YCC330 C. rollhanseniana UM113 C. rollhanseniana UM110 L000 IC. minima UM85

C. minima UM235 Ceratocystiopsis sp. MAE6P-8-4-C1 Ceratocystiopsis sp. MBE6P-3-5-C1 Ceratocystiopsis sp. WY21TX1-2 Ceratocystiopsis sp. WY21TX2-2 Ceratocystiopsis sp. WY13TX1-3

100/100 Lar C. ranaculosa CBS 216.88

100/100 ce

78/-___400/100 I

—

C. ranaculosa C1245 97/96 C. brevicomis UM1452

100/- 100/100|C: brevicomis CQ05 C. brevicomis GO05

moe C. pallidobrunnea WIN (M) 51

C. synnemata KFL17718DA 100/100°C. synnemata KFL16918DA H 100/100 C. lunata CMW55897 T C. lunata CMW55898

C. collifera CBS 126.89 Ophiostoma bacillisporum MUCL 44874 T

H 0.02

Figure |. Maximum likelihood phylogeny of Ceratocystiopsis using complete ITS and partial BT gene re- gions. The isolates recovered in this study are highlighted in color and in bold font. ML and MP bootstrap support values > 75 are indicated at the nodes as ML/MP. Bold branches indicate posterior probabilities

values = 0.9. T indicates ex-type cultures.

188 Runlei Chang et al. / MycoKeys 83: 181—208 (2021)

98/78 CC. vant ier = SNM Tt o LSU AS aN ee

C. ranaculosa CMW 13940 C. ranaculosa CBS 216.88 C. colliferan CBS 126.89 C. brevicomis UM1452 C. brevicomis CBS137839 Ceratocystiopsis sp. WY13TX1-3 C. manitobensis UM237 400/99 C. manitobensis UM214

C. minuta-bicolor CBS 635.66 C. minuta-bicolor UM844 C. rollhanseniana UM113 C. rollhanseniana UM110

C. minuta CBS 116796 99/95 C. minuta UM1532 T Ceratocystiopsis sp. YCC329

C. concentrica WIN(M)71-07 Ceratocystiopsis sp. YCC330 C. minima UM235 100/100 C. minima UM85 C. pallidobrunnea UM51

Ophiostoma bacillisporum CBS 771.71 T H 0.002

Figure 2. Maximum likelihood phylogeny of Ceratocystiopsis using partial LSU gene regions. ‘The isolates recovered in this study are highlighted in color and in bold font. ML and MP bootstrap support val- ues > 75 are indicated at the nodes as ML/MP. Bold branches indicate posterior probabilities values > 0.9.

T indicates ex-type cultures.

Species resided in the O. ips species complex were analyzed using ITS and BT gene regions. In the ITS and BT trees, our isolates of Taxon 6 (141 isolates) formed mono- phyletic clades with O. ips (Suppl. material 2).

Isolates from the S. gossypina species complex were analyzed using ITS, BT, and CAL gene regions. In the phylogenetic analyses, our isolates of Tax- on 7 were found to be closely related to two fungal isolates from China that were

previously identified as S. cf abietina (Figs 6-8).

Taxonomy

1. Ceratocystiopsis yantaiensis R.L. Chang & X.Y. Zhang, sp. nov. Fig. 9 MycoBank No: 839252

Holotype. Cuina. Shandong province: Kunyushan National Forest Park, Yantai city, from the gallery of Cryphalus piceae on Pinus thunbergii, 2 Sep. 2020, R. L. Chang (HMAS249924-holotype; SNM650 = CGMCC3.20247 — ex-holotype culture).

Additional cultures checked. Cuina. Shandong province: Kunyushan National Forest Park, Yantai city, from the gallery of Cryphalus piceae on Pinus thunbergii, 2 Sep. 2020, R. L. Chang (SNM582).

Etymology. The name refers to Yantai City, where this fungus was isolated.

Diagnosis. Ceratocystiopsis yantaiensis differs from closely related species by the production of smaller conidia.

Ophiostomatoid fungi associated with Cryphalus piceae in China 189

Gr. translucens SNM104 ITS Gr. translucens SNM101 Taxon 3 Gr. translucens SNM144 T 90/96 |Gr. anningense CXY1939 T 99/99 r. anningense CXY1944 Gr. anningense CXY1940 Gr. puerense CMW41667 Gr. puerense CMW41942 acuminatum CBS 145811 Gr. acuminatum CBS 145809 91/8 Gr. acuminatum CBS 145825 Gr. acuminatum CBS 145828 T Gr. acuminatum CBS 145827 Gr. acuminatum CBS 145810 Gr. acuminatum 110bPRJ = ‘4 : : Gr. niveum SNM145 T os / Gr. niveumSNM100_ Taxon Gr. crescericum N2015 0763 2 1 76/- |Gr. crescericum CBS 145823 Gr. crescericum CBS 145820 Gr. crescericum CBS 145821 81/90 Gr. crescericum CBS 145822 Gr. crescericum CBS 145824 Gr. crescericum 17114aFJD 92/94 ||Gr. sexdentatum N2015-1067/1/4 Gr. sexdentatum N2015-1087/2/1 Gr. sexdentatum CBS 145814 T 90/91] |Gr. crescericum CBS 130866 Gr. crescericum CBS 130864 -/86 | |Gr. furuicola CBS 145836 Gr. furuicola CBS 145812 100/100 | Gr. furuicola CBS 145813 T aml Gr. jpis-grandicollis VPRI43761 Gr. ipis-grandicollis VPRI43762 T 100/100 187/-- Gr ‘kesivae CMW41657 Gr. kesivae CMW41729 84/- 99/99 Gr. interstitiale CBS 145818 Gr. interstitiale CBS 145816 T 89/99 'Gr. interstitiale CBS 145817 100/100|Gr. roseum T46 Gr. roseum T79 Gr. roseum T72 100/99 Gr.microcarpum YCC459 Gr.fragrans CBS 279 54 T 100/99" Gr. fragrans 44aMFJD 100/100 Gr. fragrans 9214FJD Gr. sparsum CBS 405 77 T 87/ Gr. sparsum N2015-0869/2/2 “Gr. sparsum CMW54772 99/100 Gr. gorcense CMW34153 T Gr. gorcense CMW34151 Gr. gorcense CMW34152 Gr. carpaticum 132aMFJD Gr. carpaticum 143aMFJD T Gr. carpaticum CBS 145837 Gr. rectangulosporium TFM FPH 7756 T Gr. tsugae UAMH 11701 Gr. nigrum CBS 163.61 Gr. curvidentis 17814aFJD 98/100 Gr. curvidentis 55KFJD T Sporothrix dentifunda CMW 13016 100/100 Sporothrix dentifunda KFL408DB16bRJ

9) Re

4

0.1

Figure 3. Maximum likelihood phylogeny of Graphilbum using complete ITS region. The isolates recovered in this study are highlighted in color and in bold font. ML and MP bootstrap support values > 75 are indicated at the nodes as ML/MP. Bold branches indicate posterior probabilities values > 0.9. T indicates ex-type cultures.

Description. Sexual morph is unknown. Asexual state hyalorhinocladiella-like: the conidiophores directly arising singly from the vegetative hyphae, measuring (2.4—) 4.7-26.7 (46.4) um x (0.8—) 1.0-1.5 (-1.8) pm (Fig. 9d, e); or a short basal cell which continues to develop short lateral and terminal extensions from conidiogenous sites at their apices or discrete basal cells that produce 1-5 branches, which then branch irregularly and form conidiogenous cells at their apices, measuring (12.2—)

190 Runlei Chang et al. / MycoKeys 83: 181—208 (2021)

Gr. acuminatum CBS 145827 Gr. acuminatum CBS 145825 Gr. acuminatum CBS 145811 400/99 |Gr. acuminatum CBS 145828 T Gr. acuminatum CBS 145810 Gr. acuminatum CBS 145826 Gr. acuminatum CBS 145809 Gr. anningense CXY1939 T Gr. anningense CXY1940 87/88 Gr. anningense CXY 1944 : Gr. translucens SNM104 82/83 Gr. translucens SNM144 T Taxon 3 Gr. translucens SNM101 Gr. puerense CMW41619 Gr. puerense CMW41998 Gr. puerense CMW41971 95/100} Gr. puerense CMW41996 Gr. puerense CMW41671 Gr. puerense CMW41942 T 78/98 Gr. puerense CMW41667 Gr. puerense CMW41673 Gr. sparsum CBS 405.77 T Gr. fragrans 44aMFJD 100/100 Gr. fragrans 9214FJD 100/100|Gr. carpaticum 132aMFJD 77/- Gr. carpaticum 143aMFJD T Gr. carpaticum CBS 145837 97/82 |Gr. gorcense CMW34151 400/100 Gr. gorcense CMW34152 400/100 Gr. gorcense CMW34153 T Gr. ipis-grandicollis VPR|I43761 Gr. iis arandigons. SNMA45 7 T 7 or. niveum Taxon 4 . 100/100 | Gr. niveum SNM100 Taxon s | 7 Gr. kesiyae CMW41729 T 100/100" Gr. kesiyae CMW41774 Sporothrix dentifunda CMW13016 a, 100/100 Sporothrix dentifunda KFL408DB16bRJ

BT

100/100|Gr. carpaticum CBS 145837 95/94 Gr. carpaticum CBS 145835 T Gr. carpaticum CBS 145834

Gr. curvidense KFL17814aFJD 95/99 99/100°Gr. curvidense CBS 145832 T

Gr. acuminatum CBS 145825

99/100 a Gr. acuminatum CBS 145828 T Gr. acuminatum CBS 145827 95/100 || Gr. translucens SNM101

CAL

98/99] Gr. translucens SNM144 T Taxon 3 Gr. translucens SNM104 Gr. crescericum CBS 145822 Gr. crescericum CBS 145821 Gr. crescericum CBS 145823 Gr. crescericum N2015-0763/2/1 98/76 HGr. crescericum CBS 130864 Gr. crescericum CBS 130866 94/97] \Gr. crescericum CBS 145820 | | Gr. crescericum CBS 145824 7 - 99/85 Gr. niveum SNM100 _ “7 100/100 Gr. niveum SNM145 T Taxon 4

93/89 Gr. sexdentatum CBS 145815 100/100 Gr. sexdentatum 2015-1067/1/4 100/100 Gr. sexdentatum CBS 145814 T 90/- Gr. furuicola CBS 145813 T 97/100/Gr. furuicola CBS 145836 r. furuicola CBS 145812 100/94 99/10 Gr. interstitialis CBS 145818 Gr. interstitialis CBS 145817 78/82 400/100 Gr. interstitialis CBS 145816 T Gr. ipis-grandicollis VPRI43761 Gr. ipis-grandicollis VPRI43762 T 400/100 MN549015 Gr. sparsum CBS 405.77 T Gr. fragrans CMW44159

Gr. fragrans CMW43200 100/100 Snorothrix dentifunda KFL408DB16bRJ

Sporothrix dentifunda CBS 115790 T

-———1 0.2

Figure 4. Maximum likelihood phylogeny of Graphilbum using partial BT and partial CAL gene regions. The isolates recovered in this study are highlighted in color and in bold font. ML and MP bootstrap support values > 75 are indicated at the nodes as ML/MP. Bold branches indicate posterior probabilities

values > 0.9. T indicates ex-type cultures.

Ophiostomatoid fungi associated with Cryphalus piceae in China 191

EF Gr. acuminatum CBS 145827 Gr. acuminatum CBS 145825 Gr. acuminatum CBS 145828 T -(99 {Gr. acuminatum CBS 145811 Gr. acuminatum 110bPRJ Gr. acuminatum CBS 145809 91/100 Gr. acuminatum CBS 145810 Gr. translucens SNM144 T 400/100 ra Gr. translucens SNM104 Taxon 3 Gr. translucens SNM101 Gr. puerense CMW41667 Gr. puerense CMW41619 98/84 7] Gr. puerense CMW41998 Gr. puerense CMW41673 93/94 F Gr puerense CMW41671 Gr. puerense CMW41971 98/100 Gr. puerense CMW41996

Cispustease CM es aT. Gr. crescericum CBS 145820

Gr. crescericum CBS 145824 Gr. crescericum CBS 145822 84/89]Gr. crescericum CBS 145823 92/86 Gr. crescericum N2015-0763/2/1 Gr. crescericum CBS 145821 -/100| Gr crescericum 17114aFJD mamst!|Gr. crescericum CBS 130864 100/99 Gr. crescericum CBS 130866 88/5) | Gr. niveum SNM145T Taxon 4 95/967 | Gr.niveumSNM100 ab a a 99/97| lut00/100)Gr. sexdentatum CBS 145815 Gr. sexdentatum CBS 145814 T Gr. sexdentatum strain I. Gr. furuicola CBS 145812 100/100 Gr. furuicola CBS 145813 T Gr. furuicola CBS 145836 100/1007 Gr. kesiyae CMW41729 100/99 100/100) * Gr. kesiyae CMW41657 Gr. interstitiale CBS 145818 100/98\G" interstitiale CBS 145817 Gr. interstitiale CBS 145816 T Gr. ipis-grandicollis VPRI43761 100/100 Gr. ipis-grandicollis VPRI43762 T Gr. fragrans 44aMFJD 100/100 Gr. fragrans 9214FJD 100/100 |Gr. carpaticum CBS 145834 Gr. carpaticum CBS 145837 Gr. carpaticum CBS 145835 T

100/400 Gr. curvidentis 55KFJD T Gr. curvidentis 17814aFJD 100/100 Gr. gorcense CMW34153 T Gr. gorcense CMW34151 Gr. gorcense CMW34152 100/100 Gr. sparsum CBS 405.77 T

Gr. sparsum N2015-0869/2/2 98/94 Gr. sparsum CMW54772 Sporothrix dentifunda KFL408DB16bRJ

rH

0.05

Figure 5. Maximum likelihood phylogeny of Graphilbum using partial EF gene region. ‘The isolates re- covered in this study are highlighted in color and in bold font. ML and MP bootstrap support values > 75 are indicated at the nodes as ML/MP. Bold branches indicate posterior probabilities values > 0.9. T indi- cates ex-type cultures.

6.2—10.2 (—50.7) um long (Fig. 9b, c); conidiogenous cells measuring (4.7—) 6.2—10.2 (—12.4) x (0.7—) 0.9-1.3(-1.5) um (Fig. 9b, c); conidia hyaline, smooth, unicellular, short oblong, with rounded ends, measuring (1.1—) 1.4—2.2 (—2.7) x (0.8—) 0.9-1.2 (—1.5) um (Fig. 9b-e).

Culture characteristics. The Colonies are light brown in color on MEA (Fig. 9a). Mycelia are white, superficially growing on the agar. The optimal temperature for growth was 30-35 °C, reaching 43.0 mm diam in 10 days. No growth was observed at 5 °C.

192 Runlei Chang et al. / MycoKeys 83: 181—208 (2021)

ITS

S. cf. abietina CMW 109

S. cf. abietina CMW110

S. fusiformis CMW8281

87/-Js. fusiformis CMW9968 T S. fusiformis CMW8285 S. fusiformis CMW10565

ll

94/88 79/76

S. cracoviensis CBS147941 S. cracoviensis CBS147942 T S. lunata CMW10563 T S. lunata CMW 10564 S. prolifera CBS 251 88 T S. prolifera KFL218N16TARAO S. euskadiensis CMW27898 S. euskadiensis CMW27318 T S. euskadiensis CMW27899 S. rossii CBS 116 78 T 400/99 S. cantabriensis CMW39768 S. cantabriensis CMW39766 T 95/97 ey S. cantabriensis CMW39767 . Cf. abietina CMW26269 S. cf. abietina CMW 26262 S. abietina CBS125 89 T S. gossypina ATCC 18999 T 99 99>: curviconia 2 CMW17163 S. fraxini CBS147936 T S. fraxini CBS147938 100/100 S. variecibatus CMW23051 T 97/95 S. variecibatus CMW23060 |S. uta CMW40318 T S. uta CMW40316 S. uta CMW40317 g7/- 100/100 S. eucastanea CBS 424 77 T — S. eucastanea KFL1141N16DBRJ S. aurorae CMW 19362 T 100/99 S. aurorae CMW 19363 100/100 S. nebularis CMW27319 T S. nebularis CMW27900

96/94

91/75

—_ 0.005

Figure 6. Maximum likelihood phylogeny of Sporothrix gossypina species complex using complete ITS region. The isolates recovered in this study are highlighted in color and in bold font. ML and MP boot- strap support values > 75 are indicated at the nodes as ML/MP. Bold branches indicate posterior prob-

abilities values > 0.9. T indicates ex-type cultures.

Distribution. Currently known from Yantai City in Shandong Province, China.

Note. Ceratocystiopsis yantaiensis is phylogenetically close to C. manitobensis, but formed a distinct clade on both ITS, LSU, and BT trees (Figs 1 and 2). Two types of hyalorhinocladiella-like asexual state were also observed in C. manitobensis (Hausner et al. 2003). Conidia of C. yantaiensis and C. manitobensis are similar in morphol- ogy, but there is a difference in size (1.1-2.7 x 0.8-1.5 vs. 3.0-5.5 x 1.0—2.0 um, Fig. 9b-e).

2. Ceratocystiopsis wethaiensis R.L. Chang & X.Y. Zhang, sp. nov. Fig. 10 MycoBank No: 839253

Holotype. Cuina. Shandong province: Zhujiajuan village, Huancui District, Weihai City, from the gallery of Cryphalus piceae on Pinus thunbergii, 2 Sep. 2019, R. L. Chang (HMAS 249923-holotype; SNM649 = CGMCC3.20246 — ex-holotype culture).

Ophiostomatoid fungi associated with Cryphalus piceae in China 193

S. vies ane Be a BT S. villosa SNM188 T ones? -I79] §. villosa SNM182 Taxgte S. villosa SNM185 S. cf. abietina CMW110 S. cf. abietina CMW109 S. abietina CMW22310 T 95/95 S. cantabriensis CMW39768 S. cantabriensis CMW39766 T i S. cantabriensis CMW39767 95/83--+Hel S. Junata CMW 10563 | S. lunata CMW 10564 93/87 S. cracoviensis CBS147941 | S. cracoviensis CBS147942 T S. rossii CBS 116.78 T 100/851 <” rolifera CBS 251.88 T S. prolifera KFL218N16TARAO S. curviconia 2 CBS 541.84 S. fusiformis CMW10565 77/78 S. fusiformis CMW 9968 S. fusiformis CMW8281 S. fusiformis CMW8285 S. gossypina ATCC 18999 90/75\\| |S. cf. abietina CMW26262 S. cf. abietina C201 S. cf. abietina CMW26269 S. cf. abietina CMW 1468 S. cf. abietina DLS1340 S. euskadiensis CMW27318 T S. abietina NZFS 1556 S. euskadiensis CMW27899 S. abietina KUC 2671 S. euskadiensis CMW27898

100/99_s. variecibatus CMW23051 T S. variecibatus CMW23060 S. fraxini CBS147936 T 89/98 _'S. fraxini CBS147938 100/87 ~~ 995 eucastanea KFL1141N16DBRJ S. eucastanea CBS 424.77 T S. aurorae CMW 19363 99/97'S. aurorae CMW19362 T

S. uta CMW40316 S. uta CMW40317

S. uta CMW40318 T 100/100 S. nebulare CMW 27900

H S. nebulare CMW 27319 0.01

Figure 7. Maximum likelihood phylogeny of Sporothrix gossypina species complex using partial BT gene region. The isolates recovered in this study are highlighted in color and in bold font. ML and MP boot- strap support values > 75 are indicated at the nodes as ML/MP. Bold branches indicate posterior prob-

abilities values > 0.9. T indicates ex-type cultures.

Additional cultures checked. Cutna. Shandong province: Zhujiajuan village, Huancui District, Weihai City, from the gallery of Cryphalus piceae on Pinus thunber- gii, 2 Sep. 2019, R. L. Chang (SNM634).

Etymology. The name refers to Weihai City, where this fungus was isolated.

Diagnosis. Compared to other closely related species, the conidia of C. weihaiensis are smaller.

Description. Sexual morph is unknown. Asexual state hyalorhinocladiella-like: the conidiophores directly arise singly from the vegetative hyphae, measuring (2.6—) 10.9— 29.2 (44.6) um x (0.7—) 0.9-1.3 (-1.6) um (Fig. 10b-e); conidia hyaline, smooth, unicellular short oblong, with rounded ends or clavate, ellipsoidal to ovoid measuring

(1.5—) 2.0-2.6 (—2.9) x (0.7—) 0.9-1.2 (-1.5) um (Fig. 10b-e).

194 Runlei Chang et al. / MycoKeys 83: 181-208 (2021)

Ss. ee a oe S. villosa SNM185 oh OS. villosa SNM182 HED go ck |S. villosa SNM188 T 82/81 4S. cf. abietina CMW26262 S. cf. abietina CMW26269 S. abietina CMW22310 T S. curviconia CMW17163 T S. gossypina ATCC 18999 T S. euskadiensis CMW27318 T S. euskadiensis CMW27898 S. euskadiensis CMW27899 S. cantabriensis CMW39768 S. cantabriensis CMW39767 S. cantabriensis CMW39766 T

i | S.cracoviensis CBS147941

CAL

100/100--—

S.cracoviensis CBS147942 T S./unata CMW 10563 T . prolifera KFL218N16TARAO S. prolifera CBS 251.88 T S. fusiformis CMW7131 S. fusiformis CMW9968 T S. fusiformis KFL43916RJSR 400/- S. uta CMW40317 S. uta CMW40318 T 89/- S. uta CMW40316 S. aurorae CBS 118837 T 100/100 S. eucastanea CBS 424.77 100/92 S. eucastanea KFL1141N16DBRJ S. variecibatus CBS 121960 S. variecibatus CBS 121961 T S. fraxini CBS147936 T 100/100 S. fraxini CBS147938 100/100 S. nebularis CMW27319

4 S. nebularis CMW27900 0.01

100/92

Figure 8. Maximum likelihood phylogeny of Sporothrix gossypina species complex using partial CAL gene region. The isolates recovered in this study are highlighted in color and in bold font. ML and MP bootstrap support values = 75 are indicated at the nodes as ML/MP. Bold branches indicate posterior probabilities values > 0.9. T indicates ex-type cultures.

Culture characteristics. The colonies are light brown in color on MEA (Fig. 10a). Mycelia white, submerged in the agar. The optimal temperature for growth is 30 °C, reaching 46.0 mm diam in 10 days. Growth is slower at 35 °C, 27 mm diam in 10 days.

Distribution. Currently known from Weihai City in Shandong Province, China.

Note. Ceratocystiopsis weihaiensis is phylogenetically close to C. minuta, but formed a distinct monophyletic clade on both ITS and BT trees (Figs 1 and 2). In the phy- logenetic study of C. minuta by Plattner et al. (2009) using ITS, LSU, and BT gene regions, the authors suggested that this taxon is possibly an assemblage of multiple species. Therefore, they designated the strain RJ705 from Poland as the neotype. Later, strain RJ705 = UAMH 11218 = WIN(M) 1532 was considered as the lectotype for C. minuta (Reid and Hausner 2010).

Ceratocystiopsis minuta and most other Ceratocystiopsis species have a hyalorhino- cladiella-like asexual state (Plattner et al. 2009; De Beer and Wingfield 2013). The conidia of C. weihaiensis and C. minuta are similar in gross morphology. The C. wei- haiensis differs from C. minuta in having short conidia size (1.5—2.9 x 0.7-1.5 vs. 2-4 x 1-2 um, Fig. 10b-e) (Reid and Hausner 2010).

Ophiostomatoid fungi associated with Cryphalus piceae in China 195

Figure 9. Morphological characters of asexual structures of Ceratocystiopsis yantaiensis sp. nov. a fourteen- day-old culture on MEA b, € type 1 conidiophores and conidia d-e type 2 conidiophores and conidia. — Scale bars: 10 pm.

3. Graphilbum translucens R.L. Chang & X.Y. Zhang, sp. nov. Fig. 11 MycoBank No: 839254

Holotype. Cuina. Shandong province: Zhujiajuan village, Huancui District, Weihai City, from the gallery of Cryphalus piceae on Pinus thunbergii, 10 Oct. 2019, R. L. Chang (HMAS 249925-holotype; SNM144 = CGMCC 3.20263 — ex-holotype culture).

Additional cultures checked. Cutna. Shandong province: Laojiangou village, La- oshan District, Qingdao City, from the gallery of Cryphalus piceae on Pinus densiflora, 2, Aug. 2020, R. L. Chang (SNM104).

Etymology. The name refers to the translucent appearance of the colony on MEA.

Diagnosis. Graphilbum translucens can be distinguished from other closely related species, Gr. puerense and Gr. acuminatum, by the shorter hyalorhinocladiella-like con- idiophores, smaller conidia and no pesotum-like asexual state.

Description. Sexual morph is unknown. Asexual state hyalorhinocladiella-like: the conidiophores directly arising from the vegetative hyphae, measuring (3.6—) 8.6— 42.2 (—72.3) um x (0.9—) 1.1-1.7 (—2.0) um (Fig. 11b-e); conidia hyaline, smooth, unicellular short oblong, with rounded ends or ellipsoidal to ovoid, measuring (2.1-) 2.4-3.5 (4.1) x (0.8-) 1.3-2.0 (2.7) pm (Fig. 11b-e).

Culture characteristics. The colonies are light brown in color on MEA (Fig. 11a). Mycelia are partially submerged in the agar. The optimal temperature for growth is 30 °C, reaching 74.0 mm diam in 5 days. Growth slower at 35°C, 24 mm diam in 5 days. No growth was observed at 5 °C.

196 Runlei Chang et al. / MycoKeys 83: 181-208 (2021)

Figure 10. Morphological characters of asexual structures of Ceratocystiopsis weihaiensis sp. nov a four- teen-day-old culture on MEA b-e conidiophores and conidia. — Scale bars: 10 ym.

Figure | 1. Morphological characters of asexual structures of Graphilbum translucens sp. nov. a fourteen-

day-old culture on MEA b-e conidiophores and conidia. — Scale bars: 10 pm.

Ophiostomatoid fungi associated with Cryphalus piceae in China 197

Distribution. Currently known from Qingdao City and Weihai City in Shandong Province, China.

Note. Based on morphology coupled with single-gene (ITS, EF, BT, and CAL) phylogenies, Graphilbum translucens is phylogenetically close to Gr puerense and Gr. acuminatum. \n the ITS tree, Gr. translucens grouped with Gr. puerense (Fig. 3) and Gr. acuminatum whereas it formed distinct clades in the BT and EF trees (Figs 4 and 5). The hyalorhinocladiella-like asexual state was observed in Gr. translucens and Gr. puerense, but it is absent in Gr. acuminatum (Chang et al. 2017; Jankowiak et al. 2020). The conidiophores of Gr. translucens are shorter than the Gr. puerense (Chang et al. 2017). Conidia of Gr. translucens and Gr. puerense form hyalorhinocladiella-like asexual states that are similar in shape, yet the conidia size of Gv translucens is smaller than Gr. puerense (2.1-4.1 x 0.8-2.7 vs. 3.5-12 x 1-3 um, Fig. 11b-e) (Chang et al. 2017). Unlike Gr puerense and Gr. acuminatum, a pesotum-like asexual state was not observed among the isolates of Gr. translucens recovered in this study.

4. Graphilbum niveum R.L. Chang & X.Y. Zhang, sp. nov. Fig. 12 MycoBank No: 840197

Holotype. Cutna. Shandong province: Zhujiajuan village, Huancui District, Weihai City, from Cryphalus piceae on Pinus thunbergii, 10 Oct. 2019, R. L. Chang (HMAS 350268-holotype; SNM145 = CGMCC3.20423-— ex-holotype culture).

Additional cultures checked. Cutna. Shandong province: Laojiangou village, La- oshan District, Qingdao City, from the gallery of Cryphalus piceae on Pinus densiflora, 2, Aug. 2020, R. L. Chang (SNM100).

Etymology. The name refers to the white mycelia that appear on the MEA after 14 days.

Diagnosis. Graphilbum niveum differs from the closely related species Gr. cresceri- cum by its shorter conidiophore and conidia.

Description. Sexual morph is unknown. Asexual state hyalorhinocladiella-like: the conidiophores directly arising from the vegetative hyphae, or produce 1—3 branches, which then branch irregularly and form conidiogenous cells at their apices, measuring (14.0—) 21.7-36.7 (—56.0) um (Fig. 12c-e); conidiogenous cell hyaline, discrete, meas- uring (6.2—)8.4-13.8 (—18.7) pm x (0.7—) 0.9-1.3 (-1.8) wm (Fig. 12c -e); conidia hyaline, smooth, unicellular oblong to ovoid, with rounded ends, measuring (2.2-) 2.6-3.4 (-4.1) x (0.8-) 1.0-1.6 (-1.8) pm (Fig. 12b-e).

Culture characteristics. Colonies at first translucent to light brown in color on MEA (7 days). Thereafter, turning white in colour after 14 days (Fig. 12a). Mycelia are partially submerged in the agar. The optimal temperature for growth is 25 °C, reach- ing 61.0 mm diam in 8 days. The growth is relatively slower at 5 and 35 °C, reaching 2.7 mm and 9.1 mm diam in 8 days, respectively.

198 Runlei Chang et al. / MycoKeys 83: 181—208 (2021)

a * ee

i cue Sake \ eo = SSA i er Shoe ee “4 >a EE Ea

Figure 12. Morphological characters of asexual structures of Graphilbum niveum sp. nov. a left: seven-

day-old culture on MEA; right: twenty-day-old culture on MEA b Conidia c-e conidiophores and co- nidia. — Scale bars: 10 pm.

Distribution. Currently known from Qingdao and Weihai City in Shandong Province, China.

Note. Phylogenetic analyses based on each ITS, EF, and CAL tree shows that Gr. niveum is phylogenetically close to Gr. crescericum (Figs 3-5). In the ITS tree (Fig. 3), Gr. niveum clustered with Gr. crescericum whereas they a distinct clade in the EF and CAL trees (Figs 4 and 5). In both these species, the asexual structure is hyalorhinocladiella-like. Nonetheless, the conidiophore of Gr. niveum is shorter than Gr. crescericum (14.0-56.0 vs. 16.3-69.9 um) (Romén et al. 2014b). Ad- ditionally, the conidia of Gr niveum and Gr. crescericum are similar in shape, but differ in sizes. The conidia of Gr. niveum (2.2—4.1 x 0.8-1.8 pm) are substantially smaller than those of G7. crescericum (4.4-6.2 x 1.7-3.3 um). Furthermore, the colony color of Gr. niveum is light brown at first, whereas that of Gr. crescericum is white (Roméon et al. 2014b).

Graphilbum niveum emerged as a sister to Gr. kesiyae in the BT tree. This is be- cause sequences for the BT gene region were unavailable for Gr. crescericum. Graphil- bum kesiyae has both pesotum-like and hyalorhinocladiella-like asexual states, whereas Gr. niveum exclusively has the latter one. Furthermore, Gr niveum’s conidiogenous cells and conidia are smaller than those of Gr. kesiyae (Chang et al. 2017).

Ophiostomatoid fungi associated with Cryphalus piceae in China 199

7. Sporothrix villosa R.L. Chang & X.Y. Zhang, sp. nov. Fig. 13 MycoBank No: 839255

Holotype. Cuina. Shandong province: Zhujiajuan village, Huancui District, Weihai City, from Cryphalus piceae on Pinus thunbergii, 10 Oct. 2019, R. L. Chang (HMAS 249926-holotype; SNM188 = CGMCC 3.20264— ex-holotype culture).

Additional cultures checked. Cutna. Shandong province: Zhujiajuan village, Huancui District, Weihai City, from Cryphalus piceae on Pinus thunbergii, 10 Oct. 2019, R. L. Chang (SNM162); Cutna. Shandong province: Zhujiajuan village, Huan- cui District, Weihai City, from Cryphalus piceae on Pinus thunbergii, 10 Oct. 2019, R. L. Chang (SNM182).

Etymology. The name refers to the velvety colony morphology of this fungus on MEA.

Diagnosis. Sporothrix villosa differ from S. abietina by the production of smaller conidia and slow growth rate on MEA at 35 °C.

Description. Sexual morph is unknown. Asexual state sporothrix-like: the conidi- ophores directly arising from the vegetative hyphae, measuring (3.2—) 6.8—23.8 (—53.6) um x (0.5—) 0.8-1.3 (-1.5) um (Fig. 13b, d and e); conidia hyaline, smooth, unicel- lular oblong to ovoid, with rounded ends, measuring (1.2—) 1.8—2.6 (—4.1) x (0.7-) 0.8—1.1 (—1.4) um (Fig. 13c).

Culture characteristics. The colonies are white in color on MEA. Mycelia were submerged in the agar. The optimal temperature for growth is 25 °C, reaching 21.1 mm diam in 10 d. Growth is extremely slow at 35°C 3 mm diam in 10 days. No growth was observed at 5 °C.

Distribution. Currently known from Weihai City in Shandong Province, China.

Note. Sporothrix villosa is closely related to two fungal isolates recovered from China in CAL tree, and another two isolates recovered from the USA in ITS and BT trees, which were previously identified as S. cf: abietina. This taxon is phylogeneti- cally distinct from all other species in the S. gossypina species complex (Figs 6-8). Six et al. (2011) classified all the isolates from China, Canada, the USA, New Zealand, Korea, and South Africa that were close to the ex-type cultures on the BT tree as S. abietina. However, these selected isolates did not form a monophyletic clade. Later, in the phylogenies using BT and CAL gene-regions, these isolates of S. abietina did not cluster with the ex-type isolates of S. abietina. Therefore, these isolates were pro- visionally identified as S. cf abietina (Romén et al. 2014a; Romon et al. 2014b). Our phylogenetic analyses indicated that isolates classified as S. abietina (Six et al. 2011) plausibly included several phylogenetic distinct species. In this study, Sporothrix vil- losa recovered produced a sporothrix-like asexual morph similar to other species in the complex. Furthermore, the conidia of S. villosa (Fig. 13c) are smaller than those of S. abietina (1.2-4.1 x 0.7—1.4 vs. 4-7.5 x 1-2 um) (Marmolejo and Butin 1990). Unlike S. abietina, S. villosa can grow slowly at 35 °C.

200 Runlei Chang et al. / MycoKeys 83: 181-208 (2021)

Figure 13. Morphological characters of asexual structures of Sporothrix villosa sp. nov. a fourteen-day- old culture on MEA b-e conidiophores and conidia. — Scale bars: 10 um.

Discussion

In the present study, we collected Cryphalus piceae and their galleries from various pine forests located near Qingdao, Weihai, and Yantai cities in the Shandong province of China. From these beetles and galleries, we recovered 175 isolates of ophiostoma- toid fungi representing seven well-defined genera. These genera were Ceratocystiopsis, Graphilbum, Graphium, Ophiostoma, and Sporothrix. Based on molecular and mor- phological data, the data indicated that five of the ophiostomatoid fungal species re- covered in this study were previously undescribed. Hence, we newly described these ophiostomatoid species as C. yantaiensis, C. weihaiensis, Gr. translucens, Gr. niveum, and S. villosa.

Ophiostoma ips was one of the most frequently isolated ophiostomatoid fungi in China and this study (Lu et al. 2009; Chang et al. 2017; Wang et al. 2018; Chang et al. 2019). Across China, this fungus was also found associated with various species of mites and bark beetles (Chang et al. 2017). As reported for Sporothrix sp.1, in the sym- biotic relationship between M. alternatus-B. xylophilus-ophiostomatoid fungi, O. ips substantially influences the survival and reproduction of the other two partners (Niu et al. 2012; Zhao et al. 2013). Earlier, O. ips was also isolated from M. alternatus, but its specific function in this symbiotic relationship is still unknown (Zhao et al. 2018). Therefore, it is not unreasonable to hypothesize that this symbiotic fungus also influ- ences the life history and population of its vector and associated nematode.

Cryphalus piceae vectors diverse groups of fungi and nematodes. At least sixty fun- gal species have been found associated with this beetle. Globally, the diversity of fungi

Ophiostomatoid fungi associated with Cryphalus piceae in China 201

that are associated with C7. piceae varies greatly (Ohtaka et al. 2002a; Ohtaka et al. 2002b; Jankowiak and Kolarik 2010; Jankowiak et al. 2017a; Jankowiak and Bilanski 2018). In Europe, several Geosmithia species were found associated with C7. piceae (Jankowiak and Kolarik 2010; Kolafik and Jankowiak 2013; Jankowiak and Bilanski 2018). However, we did not recover any Geosmithia in this study. In Poland and Ja- pan, the most frequently isolated ophiostomatoid fungi derived from C7 piceae was O. piceae, Leptographium europhioides and O. subalpinum, respectively (Ohtaka et al. 2002b; Yamaoka et al. 2004; Jankowiak and Kolarik 2010). However, in our study, the dominant fungal species was O. ips. A similar trend was also reported from other ophiostomatoid fungi-bark beetle relationships, such as those with /ps typographus and Dendroctonus valens (Taerum et al. 2013; Chang et al. 2019). This data suggests that the relationship between bark beetles and their fungal associates is casual.

This shift in the diversity of ophiostomatoid fungi that are associated with bark beetles is possibly influenced by both climatic factors and host tree species. Previously, Linnakoski et al. (2016b) indicated that temperature can significantly influence the diversity of fungi that are associated with bark beetles. This is not an unreasonable hypothesis because the climatic conditions in China, Japan, and Poland are consider- ably different, which may influence the fungal diversity associated with various species of bark beetles from these regions. In China, we isolated these ophiostomatoid fungi from C7, piceae infecting pine trees, whereas in Japan and Poland, hosts included vari- ous species of Abies (Ohtaka et al. 2002a; Ohtaka et al. 2002b; Yamaoka et al. 2004; Jankowiak and Kolarik 2010). Besides climate, this difference in the host tree species could have also influenced the diversity of symbiotic fungi associated with C7. piceae.

Ophiostomatoid fungi are an enigmatic taxonomic group (De Beer et al. 2013). As reported previously and in the present study, the morphological differences between the species are often slim (De Beer and Wingfield 2013; Chang et al. 2019). Addition- ally, marker genes used for phylogenetic identification frequently vary between species complexes (Linnakoski et al. 2016a; Yin et al. 2019). Isolates of ophiostomatoid fungi recovered from C7. piceae in Japan were exclusively identified using morphological characters (Ohtaka et al. 2002a; Ohtaka et al. 2002b; Yamaoka et al. 2004). On the other hand, those from Poland were either based on ITS sequences (Jankowiak and Kolarik 2010) or ITS, LSU, BT and EF sequences (Jankowiak et al. 2017a). Therefore, the chances of misidentification are high, which can also influence the reported diver- sity of ophiostomatoid fungi associated with C7. piceae from these regions.

In the last decade, more than a hundred ophiostomatoid fungi have been reported from China. Among these, almost half were previously undescribed species (Yin et al. 2016; Chang et al. 2017; Wang et al. 2018; Chang et al. 2019; Chang et al. 2020; Wang et al. 2020). Owing to climate change, the economic damage caused by these bark beetles and nematodes has exponentially increased in China (Li 2013; Tang et al. 2021), initiating studies focusing on the biology and control of these beetles (Sun et al. 2013). These studies simultaneously cataloged the diversity of symbiotic fungi as- sociated with these beetles, influencing fungal species discovery (Sun et al. 2013; Zhao and Sun 2017).

202 Runlei Chang et al. / MycoKeys 83: 181—208 (2021)

In this study, we recovered seven species of ophiostomatoid fungi, including five previously undescribed species from the Shandong province of China. The previous study from Shandong province reported two new ophiostomatoid fungi associated with B. xylophilus and M. alternatus collected from two pine species (Wang et al. 2018). Thus far, more than 10 bark beetle species have been reported from this prov- ince (Bai 1985; Zhu et al. 1991). Prior to this study, no attempts were made to isolate ophiostomatoid fungi from the Shandong province of China. Therefore, in the future, follow-up surveys and isolations from other bark beetle species from the province will likely allow the discovery of several novel ophiostomatoid fungi.

Acknowledgments

This work is supported by the ‘Startup Fund’ awarded to Runlei Chang by Shandong Normal University. We are very grateful to Mr. Huade Zhang and Dr. Kaijian Teng for their assistance in collecting samples in Weihai and Yantai cities, Shandong province, China. The authors are grateful to the anonymous reviewers for their valuable com- ments on earlier versions of this manuscript. Prof Almuth Hammerbacher (Forestry and Agricultural Biotechnology Institute, University of Pretoria) provided substantial assistance in revising this manuscript, for which we are most grateful.

References

Altschul SE, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403-410. https://doi.org/10.1016/s0022-2836(05)80360-2

Bai J (1985) Several beetle species in Shandong provine and their control method (in Chinese). Shandong Forestry Science and Technology 54: 69-72.

Chang R, Duong T, Taerum S, Wingfield M, Zhou X, Yin M, De Beer Z (2019) Ophiostoma- toid fungi associated with the spruce bark beetle Jps typographus, including 11 new species from China. Persoonia 42: 50-74. https://doi.org/10.3767/persoonia.2019.42.03

Chang R, Duong TA, Taerum SJ, Wingfield MJ, Zhou X, de Beer ZW (2017) Ophiostomatoid fungi associated with conifer-infesting beetles and their phoretic mites in Yunnan, China. MycoKeys 28: 19-64. https://doi.org/10.3897/mycokeys.28.21758

Chang R, Duong TA, Taerum SJ, Wingfield MJ, Zhou X, de Beer ZW (2020) Ophiostomatoid fungi associated with mites phoretic on bark beetles in Qinghai, China. IMA Fungus 11: e15. https://doi.org/10.1186/s43008-020-00037-9

Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heu- ristics and parallel computing. Nature Methods 9: e772—772. https://doi.org/10.1038/ nmeth.2109

De Beer ZW, Duong TA, Wingfield MJ (2016) The divorce of Sporothrix and Ophiostoma: solu- tion to a problematic relationship. Stud Mycol 83: 165-191. http://dx.doi.org/10.1016/j. simyco.2016.07.001

Ophiostomatoid fungi associated with Cryphalus piceae in China 203

De Beer ZW, Seifert KA, Wingfield MJ (2013) The ophiostomatoid fungi: their dual position in the Sordariomycetes. In: Seifert KA, De Beer ZW, Wingfield MJ (Eds) The Ophios- tomatoid Fungi: Expanding Frontiers. CBS, Utrecht, The Netherlands, 1-19.

De Beer ZW, Wingfield MJ (2013) Emerging lineages in the Ophiostomatales. In: Seifert KA, De Beer ZW, Wingfield MJ (Eds) The Ophiostomatoid Fungi: Expanding Frontiers. CBS, Utrecht, The Netherlands, 21—46.

Duong TA, De Beer ZW, Wingfield BD, Wingfield MJ (2012) Phylogeny and taxonomy of species in the Grosmannia serpens complex. Mycologia 104: 715-732. http://dx.doi. org/10.3852/11-109

Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes-applica- tion to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113-118. https:// doi.org/10.1111/j.1365-294X.1993.tb00005.x

Glass NL, Donaldson GC (1995) Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous Ascomycetes. Applied and Environmental Microbiology 61: 1323-1330. https://doi.org/10.1128/AEM.61.4.1323-1330.1995.

Hammerbacher A, Schmidt A, Wadke N, Wright LP, Schneider B, Bohlmann J, Brand WA, Fenning TM, Gershenzon J, Paetz C (2013) A common fungal associate of the spruce bark beetle metabolizes the stilbene defenses of Norway spruce. Plant Physiology 162: 1324-1336. https://doi.org/10.1104/pp.113.218610

Hausner G, Eyjélfsdéttir GG, Reid J (2003) Three new species of Ophiostoma and notes on Cor- nuvesica falcata. Canadian Journal of Botany 81: 40-48. https://doi.org/10.1139/b03-009

Hofstetter RW, Dinkins-Bookwalter J, Davis TS, Klepzig KD (2015) Symbiotic associations of bark beetles. In: Vega FE, Hofstetter RW (Eds) Bark beetles: Biology and ecology of native and invasive species. Academic Press, San Diego, 209-245. https://doi.org/10.1016/B978- 0-12-417156-5.00006-X

Hyde K, Norphanphoun C, Maharachchikumbura S, Bhat DJ, Jones E, Bundhun D, Chen YJ, Bao D-F, Boonmee S, Calabon M, Chaiwan N, Kandawatte T, Dai D-Q, Dayarathne M, Devadatha B, Dissanayake A, Dissanayake L, Doilom M, Dong W, Zhang S (2020) Refined families of Sordariomycetes. Mycosphere 11: e1058. https://doi.org/10.5943/my- cosphere/11/1/7

Jacobs K, Bergdahl DR, Wingfield MJ, Halik S, Seifert KA, Bright DE, Wingfield BD (2004) Leptographium wingfieldii introduced into North America and found associated with exotic Tomicus piniperda and native bark beetles. Mycological Research 108: 411-418. https:// doi.org/10.1017/S0953756204009748

Jankowiak R, Bilanski P (2018) Geosmithia species associated with fir-infesting beetles in Po- land. Acta Mycologica 53 pp. https://doi.org/10.5586/am.1115

Jankowiak R, Kolarik M (2010) Fungi associated with the fir bark beetle Cryphalus piceae in Poland. Forest Pathology 40: 133-144. doi:10.1111/j.1439-0329.2009.00620.x

Jankowiak R, Solheim H, Bilariski P, Marincowitz S, Wingfield MJ (2020) Seven new species of Graphilbum from conifers in Norway, Poland, and Russia. Mycologia 112: 1-23. https:// doi.org/10.1080/00275514.2020.1778375

Jankowiak R, Strzatka B, Bilafski P, Kacprzyk M, Lukasova K, Linnakoski R, Matwiejczuk S, Misztela M, Rossa R (2017a) Diversity of Ophiostomatales species associated with conifer-

204 Runlei Chang et al. / MycoKeys 83: 181-208 (2021)

infesting beetles in the Western Carpathians. European Journal of Forest Research 136: 939-956. https://doi.org/10.1007/s10342-017-1081-0

Jankowiak R, Strzatka B, Bilariski P, Linnakoski R, Aas T, Solheim H, Groszek M, de Beer ZW (2017b) Two new Leptographium spp. reveal an emerging complex of hardwood-infecting species in the Ophiostomatales. Antonie van Leeuwenhoek 110: 1537-1553. https://doi. org/10.1007/s10482-017-0905-8

Justesen MJ, Hansen AK, Thomsen IM, Byriel DB, Ro-Poulsen H, Ravn HP (2020) Contribu- tions to the knowledge on biology and phenology of Cryphalus piceae (Coleoptera: Curcu- lionidae: Scolytinae). Scandinavian Journal of Forest Research 35: 468-475. https://doi.or g/10.1080/02827581.2020.1797868

Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30: 772-780. https://doi. org/10.1093/molbev/mst010

Klepzig KD, Six DL (2004) Bark beetle-fungal symbiosis: context dependency in complex as- sociations. Symbiosis (Rehovot) 37: 189-205.

Kolatik M, Jankowiak R (2013) Vector Affinity and Diversity of Geosmithia Fungi Living on Subcortical Insects Inhabiting Pinaceae Species in Central and Northeastern Europe. Mi- crobial Ecology 66: 682-700. https://doi.org/10.1007/s00248-013-0228-x

Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35: 1547-1549. https://doi. org/10.1093/molbev/msy096

Li Y (2013) Causes of Tomicus Piniperda L. disaster in Yuxi of Yunnan province and counter- measures of prevention and control (in Chinese). Forest Resources Management 3: 40—42.

Linnakoski R, Jankowiak R, Villari C, Kirisits T; Solheim H, de Beer ZW, Wingfield MJ (2016a) The Ophiostoma clavatum species complex: a newly defined group in the Ophios- tomatales including three novel taxa. Antonie van Leeuwenhoek 109: 987-1018. https:// doi.org/10.1007/s10482-016-0700-y

Linnakoski R, Mahilainen S, Harrington A, Vanhanen H, Eriksson M, Mehtatalo L, Pappinen A, Wingfield MJ (2016b) Seasonal succession of fungi associated with ps typographus bee- tles and their phoretic mites in an outbreak region of Finland. PloS ONE 11: e0155622. https://doi.org/10.1371/journal.pone.0155622

Lu M, Zhou XD, De Beer ZW, Wingfield MJ, Sun JH (2009) Ophiostomatoid fungi associ- ated with the invasive pine-infesting bark beetle, Dendroctonus valens, in China. Fungal Diversity 38: 133-145.

Marincowitz S, Duong TA, De Beer ZW, Wingfield MJ (2015) Cornuvesica: A little known mycophilic genus with a unique biology and unexpected new species. Fungal Biology 119: 615-630. http://dx.doi.org/10.1016/j.funbio.2015.03.007

Marmolejo J, Butin H (1990) New conifer-inhabiting species of Ophiostoma and Ceratocystiop- sis (Ascomycetes, Microascales) from Mexico. Sydowia 42: 193-199.

Michalski J, Mazur A (1999) Bark Beetles. A practical guide for foresters. (Korniki. Praktyczny przewodnik dLa lesnikéw). (in Polish). Oficyna Edytorska Wydawnictwo Swiat, Warszawa 125: 215-215. https://doi.org/10.1046/j.1439-0418.2001.0502b.x

Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for infer- ence of large phylogenetic trees. Gateway Computing Environments Workshop (GCE).

Ophiostomatoid fungi associated with Cryphalus piceae in China 205

Institute of Electrical and Electronics Engineers, New Orleans, LA, 1-8 pp. https://doi. org/10.1109/GCE.2010.5676129

Nel WJ, Wingfield MJ, de Beer ZW, Duong TA (2021) Ophiostomatalean fungi associated with wood boring beetles in South Africa including two new species. Antonie van Leeu- wenhoek. https://doi.org/10.1007/s10482-021-01548-0

Niu H, Zhao L, Lu M, Zhang S, Sun J (2012) The ratio and concentration of two monoterpe- nes mediate fecundity of the pinewood nematode and growth of its associated fungi. PloS one 7: e31716 https://doi.org/10.1371/journal.pone.0031716

Ohtaka N, Masuya H, Kaneko S, Yamaoka Y (2002a) Two Ophiostoma species associated with bark beetles in wave-regenerated Abies veitchii forests in Japan. Mycoscience 43: 0151- 0157. https://doi.org/10.1007/s102670200022

Ohtaka N, Masuya H, Kaneko S, Yamaoka Y, Ohsawa M (2002b) Ophiostomatoid fungi as- sociated with bark beetles on Abies veitchii in wave-regenerated forests. Journal of Forest Research 7: 145-151. https://doi.org/10.1007/BF02762603

Plattner A, Kim J-J, Reid J, Hausner G, Lim YW, Yamaoka Y, Breuil C (2009) Resolving taxo- nomic and phylogenetic incongruence within species Ceratocystiopsis minuta. Mycologia 101: 878-887. https://doi.org/10.3852/08-132

Reid J, Hausner G (2010) The epitypification of Ophiostoma minutum, now Ceratocystiopsis minuta. Mycotaxon 113: 463-474. https://doi.org/10.5248/113.463

Romén P, De Beer ZW, Fernandez M, Diez J, Wingfield BD, Wingfield MJ (2014a) Ophios- tomatoid fungi including two new fungal species associated with pine root-feeding beetles in northern Spain. Antonie van Leeuwenhoek 106: 1167-1184. https://doi.org/10.1007/ s10482-014-0286-1

Romén P, De Beer ZW, Zhou XD, Duong TA, Wingfield BD, Wingfield MJ (2014b) Multi- gene phylogenies of Ophiostomataceae associated with Monterey pine bark beetles in Spain reveal three new fungal species. Mycologia 106: 119-132. https://doi.org/10.3852/13-073

Ronquist E, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic in- ference and model choice across a large model space. Systematic biology 61: 539-542. https://doi.org/10.1093/sysbio/sys029

Six D, Beer ZW, Duong T, Carroll A, Wingfield M (2011) Fungal associates of the lodgepole pine beetle, Dendroctonus murrayanae. Antonie van Leeuwenhoek 100: 231-244. https:// doi.org/10.1007/s10482-011-9582-1

Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30: 1312-1313. https://doi.org/10.1093/bioinformatics/btu033

Suh DY, Hyun MW, Kim JJ, Son SY, Kim SH (2013) Ophiostoma ips from pinewood nematode vector, Japanese pine sawyer beetle (Monochamus alternatus), in Korea. Mycobiology 41: 59-62. https://doi.org/10.5941/MYCO.2013.41.1.59

Sun J, Lu M, Gillette NE, Wingfield MJ (2013) Red turpentine beetle: innocuous native becomes invasive tree killer in china. Annu Rev Entomol 58: 293-311. https://doi. org/10.1146/annurev-ento-120811-153624

Taerum SJ, Duong TA, De Beer ZW, Gillette N, Sun JH, Owen DR, Wingfield MJ (2013) Large shift in symbiont assemblage in the invasive red turpentine beetle. PloSs ONE 8: e78126. http://dx.doi.org/10.1371/journal.pone.0078126

206 Runlei Chang et al. / MycoKeys 83: 181—208 (2021)

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGAG6: molecu- lar evolutionary genetics analysis version 6.0. Mol Biol Evol 30: 2725-2729. https://doi.org/10.1093/molbev/mst197

Tang X, Yuan Y, Li X, Zhang J (2021) Maximum entropy modeling to predict the impact of climate change on pine wilt disease in China. Frontiers in Plant Science 12. https://doi. org/10.3389/fpls.2021.652500

Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically ampli- fied ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238- 4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990

Wadke N, Kandasamy D, Vogel H, Lah L, Wingfield BD, Paetz C, Wright LP, Gershenzon J, Hammerbacher A (2016) The bark-beetle-associated fungus, Endoconidiophora polonica, utilizes the phenolic defense compounds of its host as a carbon source. Plant Physiology 171: 914-931.

Wang H, Lun Y, Decock C, Lu Q, Liu H, Zhang X (2018) Ophiostomatoid fungi associ- ated with pines infected by Bursaphelenchus xylophilus and Monochamus alternatus in China, including three new species. MycoKeys 39: 1-27. https://doi.org/10.3897/mycok- eys.39.27014

Wang Z, Liu Y, Wang H, Meng X, Liu X, Decock C, Zhang X, Lu Q (2020) Ophiostoma- toid fungi associated with Jps subelongatus, including eight new species from northeastern China. IMA Fungus 11: e3. https://doi.org/10.1186/s43008-019-0025-3

White TJ, Bruns T, Lee S, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (Eds) PCR protocols: a guide to methods and applications. Academic Press, San Diego (California), 315-322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1

Wingfield MJ (1987) Fungi associated with the pine wood nematode, Bursaphelenchus Xylophi- lus, and cerambycid beetles in Wisconsin. Mycologia 79: 325-328. https://doi.org/10.108 0/00275514.1987.12025713

Wingfield MJ, Seifert KA, Webber JE Society AP (1993) Ceratocystis and Ophiostoma: tax- onomy, ecology, and pathogenicity. American Phytopathological Society,

Yamaoka Y, Masuya H, Ohtaka N, Goto H, Kaneko S, Kuroda Y (2004) Ophiostoma species associated with bark beetles infesting three Abies species in Nikko, Japan. Journal of Forest Research 9: 67-74. https://doi.org/10.1007/s10310-003-0056-9

Yin M, Wingfield MJ, Zhou X, Linnakoski R, de Beer ZW (2019) Taxonomy and phylogeny of the Leptographium olivaceum complex (Ophiostomatales, Ascomycota), including de- scriptions of six new species from China and Europe. MycoKeys 60: 93-123. https://doi. org/10.3897/mycokeys.60.39069

Yin ML, Wingfield MJ, Zhou XD, De Beer ZW (2016) Multigene phylogenies and mor- phological characterization of five new Ophiostoma spp. associated with spruce-infesting bark beetles in China. Fungal Biology 120: 454-470. http://dx.doi.org/10.1016/j.fun- bio.2015.12.004

Zhao L, Ahmad F, Lu M, Zhang W, Wickham JD, Sun J (2018) Ascarosides promote the preva- lence of ophiostomatoid fungi and an invasive pathogenic nematode, Bursaphelenchus xylophi-

lus. Journal of Chemical Ecology 44: 701-710. https://doi.org/10.1007/s10886-018-0996-3

Ophiostomatoid fungi associated with Cryphalus piceae in China 207

Zhao L, Lu M, Niu H, Fang G, Zhang S, Sun J (2013) A native fungal symbiont facilitates the prevalence and development of an invasive pathogen-native vector symbiosis. Ecology 94: 2817-2826. https://doi.org/10.1890/12-2229.1

Zhao L, Mota M, Vieira P, Butcher RA, Sun J (2014) Interspecific communication between pinewood nematode, its insect vector, and associated microbes. Trends in Parasitology 30: 299-308. nhttp://dx.doi.org/10.1016/j.pt.2014.04.007

Zhao L, Sun J (2017) Pinewood nematode Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle. In: Wan F, Jiang M, Zhan A (Eds) Biological Invasions and Its Management in China: Volume 2. Springer Singapore, Singapore, 3-21. https://doi.org/10.1007/978- 981-10-3427-5_1

Zhu C, Liu S, Lu X (1991) Species diversity and treatment of beetles in Shandong province (in Chinese). Shandong Forestry Science and Technology 2: 53-56.

Supplementary material |

Ophiostomatoid fungi associated with Cryphalus piaceae in Shandong province

in eastern China

Authors: Runlei Chang, Xiuyu Zhang, Hongli Si, Guoyan Zhao, Xiaowen Yuan,

Tengteng Liu, Tanay Bose, Meixue Dai

Data type: molecular data

Explanation note: Maximum likelihood phylogeny of Graphium using complete ITS and partial EF gene regions. The isolates recovered in this study are high- lighted in color and in bold font. ML and MP bootstrap support values > 75 are indicated at the nodes as ML/MP. Bold branches indicate posterior prob- abilities values > 0.9. T indicates ex-type cultures.

Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) 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.

Link: https://doi.org/10.3897/mycokeys.83.70925.suppl1

208 Runlei Chang et al. / MycoKeys 83: 181-208 (2021)

Supplementary material 2

Figure S2

Authors: Runlei Chang, Xiuyu Zhang, Hongli Si, Guoyan Zhao, Xiaowen Yuan,

Tengteng Liu, Tanay Bose, Meixue Dai

Data type: phylogenetic data

Explanation note: Maximum likelihood phylogeny of Ophiostoma ips species complex using complete ITS and partial BT gene regions. The isolates recovered in this study are highlighted in color and in bold font. ML and MP bootstrap support values = 75 are indicated at the nodes as ML/MP. Bold branches indicate posterior probabilities values > 0.9. T indicates ex-type cultures.

Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) 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.

Link: https://doi.org/10.3897/mycokeys.83.70925.suppl2

Supplementary material 3

Table S1

Authors: Runlei Chang, Xiuyu Zhang, Hongli Si, Guoyan Zhao, Xiaowen Yuan,

Tengteng Liu, Tanay Bose, Meixue Dai

Data type: phylogenetic data

Explanation note: List of ophiostomatioid fungi used for phylogenetic analyses. T = ex-type culture.

Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) 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.

Link: https://doi.org/10.3897/mycokeys.83.70925.suppl3