MycoKeys 50: 93- | 33 (20 | 9) A peer-reviewed open-access journal doi: 10.3897/mycokeys.50.32653 RESEARCH ARTICLE © Mycokeys http://mycokeys.pensoft.net Launched to accelerate biodiversity research Differential patterns of ophiostomatoid fungal communities associated with three sympatric Tomicus species infesting pines in south-western China, with a description of four new species Hui Min Wang!', Zheng Wang', Fu Liu', Cheng Xu Wu', Su Fang Zhang’, Xiang Bo Kong', Cony Decock’, Quan Lu', Zhen Zhang' | Key Laboratory of Forest Protection, National Forestry and Grassland Administration; Research Institu- te of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing 100091, China 2 Mycothéque de P’'Université Catholique de Louvain (BCCM/MUCL), Earth and Life Institute, Microbiology, B-1348 Louvain-la-Neuve, Belgium Corresponding author: Zhen Zhang (zhangzhen@caf.ac.cn); Quan Lu (luquan@caf.ac.cn) Academic editor: Kevin Hyde | Received 27 December 2018 | Accepted 9 March 2019 | Published 9 April 2019 Citation: Wang HM, Wang Z, Liu F, Wu CX, Zhang SF, Kong XB, Decock C, Lu Q, Zhang Z (2019) Differential patterns of ophiostomatoid fungal communities associated with three sympatric Jomicus species infesting pines in south-western China, with a description of four new species MycoKeys 50: 93-133. https://doi.org/10.3897/ mycokeys.50.32653 Abstract Bark beetles and their associated fungi, which cause forest decline and sometimes high mortality in large areas around the world, are of increasing concern in terms of forest health. Three Jomicus spp. (T brevipilosus, T: minor and. T. yunnanensis) infect branches and trunks of Pinus yunnanensis and P kesiya in Yunnan Province, in south-western China. 7omicus spp. are well known as vectors of ophiostomatoid fungi and their co-occurrence could result in serious ecological and economic impact on local forest eco- systems. Nonetheless, knowledge about their diversity, ecology, including pathogenicity and potential economic importance is still quite rudimentary. Therefore, an extensive survey of ophiostomatoid fungi associated with these Zomicus species infesting P yunnanensis and P kesiya was carried out in Yunnan. Seven hundred and seventy-two strains of ophiostomatoid fungi were isolated from the adult beetles and their galleries. The strains were identified based on comparisons of multiple DNA sequences, includ- ing the nuclear ribosomal large subunit (LSU) region, the internal transcribed spacer regions 1 and 2, together with the intervening 5.8S gene (ITS) and the partial genes of 8-tubulin (7UB2), elongation Copyright Hui Min Wang 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. 94 Hui Min Wang et al. / MycoKeys 50: 93-133 (2019) factor lox (YEFI-a) and calmodulin (CAL). Phylogenetic analyses were performed using maximum par- simony (MP) as well as maximum likelihood (ML). Combinations of culture features, morphological characters and temperature-dependent growth rates were also employed for species identification. Eleven species belonging to five genera were identified. These included six known species, Esteya vermicola, Lep- tographium yunnanense, Ophiostoma brevipilosi, O. canum, O. minus and O. tingens and four novel taxa, described as Graphilbum anningense, O. aggregatum, Sporothrix pseudoabietina and S. macroconidia. A residual strain was left unidentified as Ophiostoma sp. 1. The overall ophiostomatoid community was by far dominated by three species, representing 87.3% of the total isolates; in decreasing order, these were O. canum, O. brevipilosi and O. minus. Furthermore, the ophiostomatoid community of each beetle, al- though harbouring a diversity of ophiostomatoid species, was differentially dominated by a single fungal species; Ophiostoma canum was preferentially associated with and dominated the ophiostomatoid com- munity of 7’ minor, whereas O. brevipilosi and O. minus were exclusively associated with and dominated the ophiostomatoid communities of 7’ brevipilosus and T’ yunnanensis, respectively. Eight additional species, representing the remaining 12.7% of the total isolates, were marginal or sporadic. These results suggested that sympatric Zomicus populations are dominated by distinct species showing some level of specificity or even exclusivity. Keywords Esteya vermicola, Graphilbum, Leptographium, Ophiostoma, species-specific association, Sporothrix, taxonomy Introduction Associations between insects and microorganisms are increasingly recognised as one of the major issues in forest ecology and forest health around the world (Wingfield et al. 2016). Many bark beetles are well known as tree pests causing various levels of tree mor- tality and forest decline in large areas of the world, mostly in temperate areas (Jankow- iak 2006, Wingfield et al. 2017). These bark beetles are well known vectors of variably pathogenic fungi, forming symbiosis-like relationships (Six 2003, Lu et al. 2009). The pine shoot beetles, Zomicus Latreille (syn. Blastophagus Eichhoff, Myelophilus Eichhoff, Scolytidae, Coleoptera), are destructive insects with a range spanning the Eurasian pine forests, seriously affecting tree growth and causing a great threat to the forest ecosystems (Kirkendall et al. 2008, Lieutier et al. 2015). Currently, eight species are recorded worldwide, i.e. TZ’ armandii Li and Zhang (Li et al. 2010), 7’ brevipilosus Eggers, 7’ destruens Wollaston, 1’ minor Hartig, J pilifer Spessivtsev, 7’ piniperda L.., T. puellus Reitter, and T’ yunnanensis Kirkendall and Faccoli (Kirkendall et al. 2008). They all occur in China except 7’ destruens and five of them, viz. 7) armandii, T. brevi- pilosus, T’ minor, T: pilifer and T. yunnanensis, are sympatric in forests of the Yunnan Province (Li et al. 1997, 2010, Kirkendall et al. 2008; Ye 2011). Tomicus brevipilosus, LT. minor and T! yunnanensis have overlapping geographical distribution, host range and infection periods. They aggregately infect branches and trunks of two indigenous pines, Pinus yunnanensis and P. kesiya (Li et al. 1997, 2006, Chen et al. 2009, 2010, Lu et al. 2012, 2014), causing locally extensive tree decline or mortality (Ye and Dang 1986, Ye 1991, 2011). Since the 1980s, damage caused by these bark beetles has re- sulted in losses of more than 93,000 m° of pinewood (Ji et al. 2007). Differential patterns of ophiostomatoid fungal communities associated... 95 Generally, two or three pine shoot beetles co-occur underneath the bark or in shoots of a single host tree, either simultaneously but with spatially isolated galleries or successively, during differential infesting peaks. Spatial and chorological differentiation would reduce competition between beetles, but their co-occurrence also could enhance cooperation (Lu et al. 2012, Chen et al. 2015). Tomicus yunnanensis is considered to be the most aggressive species in Yunnan, causing primary infestations of healthy P yunnanensis trees and eventually tree death (Ye and Lieutier 1997, Kirkendall et al. 2008, Chen et al. 2010, 2015, Lu et al. 2014). Although 7° brevipilosus is able to infect healthy trees, it preferably colonises trunks already infested by 7’ yunnanensis or both TL. yunnanensis and. T: minor (Chen et al. 2010, 2015). Zomicus minor is often regarded as a secondary, opportunist species infesting trees already weakened by 7’ yunnanensis or/and 7. brevipilosus (Ye and Ding 1999, Lieutier et al. 2003, Chen et al. 2009). Pine shoot beetles such as 7) piniperda, T: minor and T: destruens are commonly as- sociated with ophiostomatoid fungi (Masuya et al. 1999, Kim et al. 2005, Jankowiak 2006, 2008). Fifteen ophiostomatoid fungi were reported associated with 7’ piniperda in Europe (Mathiesen 1950, Lieutier et al. 1989, Gibbs and Inman 1991, Solheim and Langstro6m 1991, Jankowiak 2006, Jankowiak and Bilariski 2007) and 11 were docu- mented in eastern Asia (Japan and Korea) (Masuya et al. 1999, Kim et al. 2005). Ophios- toma minus was shown to be the dominant species associated with 7’ piniperda in Europe and Japan (Mathiesen 1950, Lieutier et al. 1989, Gibbs and Inman 1991, Masuya et al. 1999, Jankowiak 2006). Leptographium wingfieldii was shown to be the strongest patho- genic one (Gibbs and Inman 1991) in Europe. Zomicus minor also infests various pines in Europe and Asia. Fifteen (Mathiesen-Kaarik 1953, Masuya et al. 1999, Jankowiak 2008) and 11 (Masuya et al. 1999) ophiostomatoid species have been reported to be associ- ated with this beetle species in Europe and Japan, respectively. Ophiostoma canum was recorded as a frequent/dominant species in association with 7’ minor, both in Europe and Japan (Mathiesen 1950, 1951, Rennerfelt 1950, Francke-Grosmann 1952, Masuya et al. 1999) but seems to represent a weak pathogen to P sylvestris (Solheim et al. 2001). Additionally, six ophiostomatoid fungi were documented associated with 7’ destruens in Europe (Lieutier 2002, Sabbatini Peverieri et al. 2006, Ben Jamaa et al. 2007). Despite the fact that 7omicus spp. have caused serious losses to forest ecosystems in south-western China, there are no systematic studies of their ophiostomatoid as- sociates but only a few sporadic reports. So far, nine ophiostomatoid species have been reported as being associated with Zomicus spp. in Yunnan. Six species (Leptographium yunnanense, Ophiostoma ips, O. minus, O. quercus, S. abietina and S. nebularis) were recorded to be associated with 7’ yunnanensis (Ye et al. 2000, Zhou et al. 2000, 2013, Chang et al. 2017). Two species (Graphilbum fragrans and O. tingens) were recorded as being associated with 7’ minor (Zhou et al. 2013, Pan et al. 2017), whereas only a sin- gle species (O. brevipilosi) was recorded as being associated with 7’ brevipilosus (Chang et al. 2017). Amongst them, L. yunnanense was the first species newly described from the area (Zhou et al. 2000) and is likely the most virulent one (Liao and Ye 2004, Gao et al. 2017). Until now, the relative abundance with which these fungi occur, their host (pine and beetle) relationships, and their pathogenicity remain unknown. 96 Hui Min Wang et al. / MycoKeys 50: 93-133 (2019) The symbiosis between bark beetles and ophiostomatoid fungi enhances their pathogenicity. The fitness of bark beetle populations may depend in part on the degree of the fungal partners’ pathogenicity and the resulting weakening of the tree (Chris- tiansen et al. 1987, Kirisits 2004, Linnakoski et al. 2012), although this has been ques- tioned by some (Six and Wingfield 2011). Therefore, the question remains whether there is any link between the differential aggression of the pine shoot beetles and the differential virulence of their fungal associates, especially in circumstances where vari- ous beetle species co-exist. The aim of this study was to describe the diversity of ophiostomatoid fungal com- munities associated with three pine shoot beetles and their galleries infesting P yunnan- ensis and P kesiya in forest ecosystems of Yunnan Province. We also analysed the degree of beetle/ophiostomatoid fungi specificity. Such studies will enable us to understand the aggressive nature of the beetles and the pathogenicity of the associated fungi and the interactions, ultimately helping to address the current situation of ceaseless out- breaks and rapid expansion of the pests. Materials and methods Sample collection and fungus isolation Samples of galleries in bark and shoots and adults of Zomicus spp. were collected from P. yunnanensis and P kesiya at five sites in Yunnan Province (Fig. 1, Table 1) from De- cember 2016 to March 2017. Beetles were placed individually in sterilised Eppendorf tubes and their galleries were placed in sterile envelopes and stored at 4°C until pro- cessed within one week. Isolations from beetles and their galleries were carried out on 2% malt extract agar (MEA: 20 g Biolab malt extract, 20 g Biolab agar and 1 000 ml deionised water) with 0.05% NaClO added, in 9-cm Petri dishes as described by Seifert et al. (1993). Hyphal tips of emerging colonies were cut and transferred to MEA plates in order to obtain pure strains. The strains were grown routinely on 2% MEA at 25 °C. Representative Table |. Basic information on the sample collection plots in China. Location Host Insect vector longitude\latitude altitude(m) No. of examained samples Xiangyun,Yunnan Pinus yunnanensis Tomicus yunnanensis, T. minor 25°21'25.8"N, 100°51'49"E 2255.4 447 Puer, Yunnan P kesiya T. brevipilosus, T. minor 22°56'36.1"N, 101°14'36.7"E 1400.7 346 Qujing, Yunnan P. yunnanensis T. yunnanensis, T! minor, 25°28'51"N, 103°46'32"E 2068.2 102 L. brevipilosus Anning, Yunnan P. yunnanensis T. yunnanensis, T. minor, 24°53'32"N, 102°24'23"E 1939.9 138 T. brevipilosus Yuxi, Yunan P. yunnanensis T. yunnanensis, T. minor 24°18'23"N, 102°34'37"E 1908.1 85 Differential patterns of ophiostomatoid fungal communities associated... way Yunnan province ¢_‘°"™ Figure |. A Map showing the 11 species of ophiostomatoid fungi detected from Yunnan Province, China B, D disease symptoms on Pinus yunnanensis and P. kesiya trees infested by Tomicus spp. (I. yun- nanensis, I: minor and T: brevipilosus) and ophiostomatoid fungi C, G, H exposed branches of Tomicus spp. on P yunnanensis and P kesiya E, F, I-K galleries of Tomicus spp. on P yunnanensis and P kesiya. cultures of each morphotype were deposited in the China Forestry Culture Collection Center (CFCC, part of the National Infrastructure of Microbial Resources) and the culture collection of the Chinese Academy of Forestry (CXY) (Table 2). Morphology and growth studies Morphological characterisation of both the sexual and asexual reproduction forms was performed on 2% MEA media incubated 3—6 weeks at 25 °C in the dark. Slide cul- tures were made to observe all microscopic characters (sexual/asexual structures) using a BX51 OLYMPUS microscope with differential interference contrast. 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CO9TOSHW LO9T6OSHW 009T6SHW 66ST6SHW 86ST6SHW 66S€89HW 86S¢89HW 96SZ69HW S6SZ69HW ¥6SLO9HW 009€89HW OL<6£09HW cEOLODHW I 70% for ML and MP are indicated above branches. Bootstrap values < 70% are indicated by the symbol *. Strains representing ex- type sequences are marked with “T’; ML, maximum likelihood; MP, maximum parsimony and the final alignment of 743 positions, including gaps. 104 Hui Min Wang et al. / MycoKeys 50: 93-133 (2019) R. sulphurea KX137173 UNVERIFIED R. sulphurea KX137175UNVERIFIED 100\100 R. montetyi EU977475 R. quercivora KX267120 R. amasae EU977470 R. sulphurea EU977467 G. francke grosmanniae AY348309 G. radiaticola AY744562 T 0.08 0.02 a b 1oo\100| G. clavigera XM014317218 G. clavigera KP171177 Figure 3. Phylograms obtained from ML analysis of 8-tubulin A and elongation factor B sequences of Esteya, showing fungal associates with pines infected by Tomicus yunnanensis in Yunnan Province, China. Novel sequences obtained in this study are printed in bold type. Bootstrap values > 70% for ML and MP are indicated above branches. Bootstrap values < 70% are indicated by the symbol *. Strains representing ex-type sequences are marked with “T’; ML, maximum likelihood; MP, maximum parsimony and the final alignment of 320 (A), 856 (B) positions, including gaps. Gra. fragrans MG205667 Gra. fragrans AF198248 T Gra. microcarpum GU134170 stool Gra. rectangulosporium AB242825 T an SECs B O. cf. rectangulosporium GU129987 CFCC52633 Gra. cf. rectangulosporium KU319038 O. cf. rectangulosporium EU785449 on1dq Gra. puerense MG205670 T Gra. puerense MG205671 CFCC52631 931001 CFCC52632 |B CFCC52633 Gra. kesivae MG205668 Gra. kesivae MG205669 T Gra sp.3. GU129997 Gra. crescericum DQ539535 Cop. manitobensis EU913714 T Gra. puerense MG205715 Gra. puerense MG205719 Gra. puerense MG205717 Gra. puerense MG205718 T Gra. puerense MG205716 Gra. kesivaee MG205713 T Si00l) Gra. kesivaee MG205714 Gra. kesivaee MG205711 Gra. kesivaee MG205712 on9ep Gra. fragrans MG205710 Gra. microcarpu KY568222 H. lignivora EF139102 100\100 Figure 4. Phylograms obtained from ML analysis of ITS sequences A and £-tubulin sequences B of Graphilbum showing fungal associates with pines infected by Tomicus yunnanensis and T: minor in Yun- nan Province, China. Novel sequences obtained in this study are printed in bold type. Bootstrap values > 70% for ML and MP are indicated above branches. Bootstrap values < 70% are indicated by the symbol *, Strains representing ex-type sequences are marked with “T’; ML, maximum likelihood; MP, maximum parsimony and the final alignment of 515 (A), 481 (B) positions, including gaps. ‘The six strains from groups D to I nested within the Ophiostoma lineage based on the LSU phylogenetic tree (Fig. 2). The ITS dataset comprised species from all line- ages discovered in this study. Analysis of this dataset yielded the phylograms shown in Fig. 6. Sixteen ITS sequences generated in this study were compared with 61 sequences Differential patterns of ophiostomatoid fungal communities associated... L. conjunctum HQ406831 T L. hindbergii DQ062068 T L.koreanum EU502798 T L. truncatum 1DQ062052 T L. albopini AF343696 T diol. pyrinum DQ062072 G, clavigera AY544613T G. aurea AY544610 T G. robusta AY 553397 T L.wingfieldii AY553398 T L, longiclavatum AY816686 T L. terebranti EU296777 T L. alethinum AY553392 L. pineti DQ062076 G. serpens AY707202 T L. neomexicanum AY553382 T L. douglasii AY553381 T L. pini densiflorae DQ062082 T L. sinoprocerum EU296773 T L. pracerum EU244638 T L. profanum DQ354944 T L. celere HQ406834 T L. bhutanense EU650187 T L. manifestum HQ406837 T ipooo) G. piceaperda AY 707209 G. europhioides AF343693 T G. elavigera complex G. wageneri complex L. procerum complex G_ piceaperda complex 105 — L. conjunetum MG205730 L. conjunctum MG205779 L. conjunctum HQ406856 L. conjunctum HQ406855 L. koreanum EU502827 L. koreanum EU502825 T L. koreanum EU502826 L.truncatum DQ062024 T L.truncatum DQ062023 §389L 7. truncatum DQ062025 L. lundbergii FJ280065 10000 IL. hindbergii DQ062033 L. lundbergii DQ062035 T L. conjunctum MG205780 r L. conjunetum MG205779 L. conjunctum HQ406856 L. conjunctum HQ406855 G, crassivaginata AF343673 T 100100 _jL. brachiatum AF343676 T L. antibioticum AF343677 T G. grandifoliae AF343711 T G. radiaticola EU502801 G. leptographioides AF343710 T LO0\100 G. dryocoetidis AF343709 T 100\100_f L. abicolens AF343701 L. hughesii AF343700 — | G. abiocarpa AF34368]1 | | G. penicillata DQ097851 T 1 | £. curviconidium HQ406848 T } |Z. altius HQ406851 T ie s L. koreanum EU502826 L. reconditum AF343689 T G. penicillata complex 7416) 'L .pinicola DQ062027 T L. abietinum AF343670 T L.truncatum DQ062024 T L.truncatum DQ062023 bgto-- guttulatum AF343683 8318! L.truneatum DQ062025 L. euphyes AF343686 T L. lundbergii FJ280065 G. americana AF343687 100100 I. lundbergii DQ062033 A geet eats L. lundbergii DQ062035 T L. fruticetum DQ097847 T 0.01 100\100_[— G. olivaceapini AJ538336 Cc G. olivacea AJ538337 a °3 Figure 5. Phylograms obtained from ML analysis of ITS2-28S A §-tubulin B and elongation factor C sequences of Leptographium, showing fungal associates with pines infected by Tomicus yunnanensis and T. brevipilosus in Yunnan Province, China. Novel sequences obtained in this study are printed in bold type. Bootstrap values = 70% for ML and MP are indicated above branches. Bootstrap values < 70% are indicat- ed by the symbol *. Strains representing ex-type sequences are marked with “T’; ML, maximum likelihood; MP, maximum parsimony and the final alignment of 641 (A), 358 (B), 639 ( C) positions, including gaps. retrieved from GenBank, representing the major groups of Ophiostoma (de Beer and Wingfield 2013, Linnakoski et al. 2016). The ITS- and 7UB2-based phylogenetic inferences (Figs 6, 7a, b) showed that the strains of groups D and E nested within the O. clavatum- and O. piceae-complex (de Beer and Wingfield 2013, Yin et al. 2016, Linnakoski et al. 2016), in which they were positioned in the near vicinity of the O. brevipilosi and O. canum clades, respectively. From these results, and considering their morphological features, we concluded that the strains of groups D and E are conspecific with O. brevipilosi and O. canum, respectively. 106 0.06 Hui Min Wang et al. / MycoKeys 50: 93-133 (2019) os\oor O. arduennense AY573241 O. distortum AY924386 O. ulmi AF198232 O.novo ulmi AF198235 ey _ O. himai ulmi AF198233 ine , sol] |O. querci AY466626 T Peat SNE Ee 95\40|'O. querci AF198239 O. catonianum AF 198243 T DOK100 O. tetropit AY194485 O. tetropii AY 194482 O. piceae AF198226 T O. piceae AF 198 O. canum HM031489 +O. ginghaiense KU184445 T O. breviusculum AB200423 T O. micans KU184432 T O. flexuosum AT924387 T O. nitidum KU184436 T O. rachisporum HM031490 T O. setosum FJ430486 24 O. setosum FJ430485 O. cupulatum AF198230 O. floccosum AF198231 T : O. macrosporum KX590821 Ambrosiella sp DQ268585 O. minus AY934509 O. minus AY542495 O. minus AM943886 O. minus AY304438 HOML001 O. minus AF234834 O. minus AY542496 tpo\100) O. olgensio KU551303 T O. olgensio KU551300 100\100)O. Aryptum AY304436 T O. kryptum AY304428 O. allantosporum AY934506 O. ainoae KU094682 T O. poligraphi KU184444 O. brevipisoli MG205660 T pn99 O. shangrilae KU184454 O. clavatum KU094685 T O. brunneo ciliatum KU094683 T s2\85] O. pseudocatenulatum KU094686 T O. brunneolum KU094684 T O. piliferum AF221070 O. clavatum complex 82\88 O. tapionis HM031493 O. bicolor DQ268604 T O. pulvinisporumAY 546714 T shel O- adjuncti AY546696 T 93\7 O. ips DQ539549 93\97 MOL O. ips AY546704 T O. fuscum HM031504 T O. japonicum GU134169 T 99100 - O- montium AY546712 O. montium AY546711 100\100 S. inflata AY495426 T S. abietina AF484453 T 96\97) O. ips complex 100\100 Figure 6. Phylograms obtained from ML analysis of ITS sequences of Ophiostoma, showing fungal asso- ciates with pines infected by Tomicus yunnanensis, T: minor and T. brevipilosus in Yunnan Province, China. Novel sequences obtained in this study are printed in bold type. Bootstrap values > 70% for ML and MP are indicated above branches. Bootstrap values < 70% are indicated by the symbol *. Strains representing ex-type sequences are marked with “T’; ML, maximum likelihood; MP, maximum parsimony and the final alignment of 633 positions, including gaps. Differential patterns of ophiostomatoid fungal communities associated... 107 98\100] O. poligraphi KU184315 T O. poligraphi KU184314 O. ainoae KU184287 O. ainoae HM031552 T O. shangrilae KU184324 T O. shangrilae KU184325 1oo00r O. tapionis KU184326 O. tapionis HM031545 T O. brunneolus KU094697 O. brunneolus HM03 1554 T O. macroclavatum KU094728 O. pseudocatenulatum KU094738 T O. pseudocatenulatum KU094734 O. brunneociliatum KU184292 O. brunneociliatum KU184293 O. clavatum KU094705 'O. clavatum KU094712 O. ips AY194955 O. minus AY 542496 O. minus AY304438 100\100] O. minus AF234834 O. minus AY542498 O. minus AY542497 North American O. minus HM031497 O. minus AY542495 O. minus AM943886 [Eurasian O. minus AY934509 O 1) DOQ53950 0. allantosporum A¥934506 100) O. kryptum AY304436 T O. kryptum AY 304428 O. olgensis KU551303 T O. piceae AF 198226 O. macrosporum KX590763 O. piliferum DQ868380 100\100_} Q. piliferum AY305705 O. piliferum AY305704 100\100) O. floccosum KU184300 O. floccosum KU184299 100\100) O. fapionis KU184326 T O. tapionis KU184327 O. araucariae KU184289 100\100 ) O. clavatum KU094705 O. clavatum KU094712 0.09 9s\100) O. nitidum KU184308 T O. nitidum KU184307 O. micans KU184303 T O. micans KU184304 O. piceae KU184313 O. piceae KU184311 ( O. piceae KU184312 4 O. breviusculum AB200428 O. breviusculum AB200427 T O. subalpinum AB200430 O. subalpinum AB200429 O. ginghaiense KU184316 T O. ginghaiense KU184317 O_rachisporum KU184319 T O. rachisporum KU184320 O. flexuosum KU184298 T O. flexuosum DQ96090 1oo100_| O. setosum KU184323 O. setosum KU184322 T 0.02 O. minus AY542505 O. minus HM03 1535 9s99|| O. minus JX444618 ‘ O. minus AY542506 Eurasian O. minus EU785430 O. minus TX046807 O. minus HM031536 O. minus HM0315364 = O. minus AY548743 O. minus AY 672913 O. minus AY542507 O. minus KC336019 aoe O. minus AY542508 | American O. minus AY542509 O. minus AY305690 CXY1936/1 10100) O. album CFCC52168 T O. album CFCC52169 O. kryptum AY305686 O. kryptum AY305685 T O. olgensis KU882942 T O. olgensis KU882938 O. piceae KU184313 d 0.02 Figure 7. Phylograms obtained from ML analysis of §$-tubulin sequences of Ophiostoma A, B, D, E and ITS sequences of O. minus-complex C showing fungal associates with pines infected by Tomicus yunnanensis, T. minor and T. brevipilosus in Yunnan Province, China. Novel sequences obtained in this study are printed in bold type. Bootstrap values > 70% for ML and MP are indicated above branches. Bootstrap values < 70% are indicated by the symbol *. Strains representing ex-type sequences are marked with “T’; ML, maximum likelihood; MP, maximum parsimony and the final alignment of 455 (A), 430 (B), 541 (C), 378 (D), 423 (E) positions, including gaps. 108 Hui Min Wang et al. / MycoKeys 50: 93-133 (2019) i. curviconia 2 KX590836 S. abietina AF484453 T S. gossypina KX590819 T |S. euskadiensis DQ674369 T . cantabriensis KF951554 T S. rossii KX590815 S. gossypina complex PAS. lunata AY280485T ‘S. prolifera KX590829 T S. fusiformis AY 280481 T S. variecibatus DQ821568 T pS: aurorae DQ396796 T S. eucastanea KX590814 T S. uta KU595577 S. stenoceras AF484475 T | eel 5: Zambiensis EU660453 T | §. stenoceras complex ~ |S. protearum AF 194510 T S. africana DQ316199 ||| S. inflata AY495426 T 900 WS. guttiliformis KX590839 T S. inflata complex S. dentifunda AY495434 T S. phasma DQ316219 T odbor| ooo SlODosa KX590838 T S. globosa KP017086 T S. luriei AB128012T Pathogenic Clade S. brasiliensis KX590832 T S. schenckii KX590842 T S. mexicana KX590841 T S. humicola AF484472 T S. pallida EF127879T S. pallida KX590831 T 83\90 S. pallida EF127880 T A|'S. stvlites EF127883 T S. pallida complex S. chilensis KP711811 T S. proteasedis EU660449 T ET) S. gemella DQ821560 T {| 'S. palmiculminata DQ316191 T S. cabralii KT362256 T WS. itsve KX590840 T soar S. candida HM05 1409 T 5. candida complex | |S. rapaneae KU595583 T S. aemulophila KT192603 T S. polyporicola KX590827 T S. inflata AY495425 95\99 |, 1q0\100. OB\86 INT . eucalyptigena KR476 sig7/9- thermara KRO51115 T zB S. epigloea KX590817 T S. bragantina FN546965 T oouno f~ S. fitmea HM051412 S. valdivianum KX590830 T S. curviconia KX590835 T deus ‘fea O. nigrocarpum AY280490 T — $. brunneoviolacea FN546959 T S. nebularis KX590824 T S. dombeyi KX590826 T 100\100 O. ips AY546698 O. bicolor DQ268604 0.08 Figure 8. Phylograms obtained from ML analysis of ITS sequences of Sporothrix, showing fungal associ- ates with pines infected by Tomicus yunnanensis, T. minor and T. brevipilosus in Yunnan Province, China. Novel sequences obtained in this study are printed in bold type. Bootstrap values > 70% for ML and MP are indicated above branches. Bootstrap values < 70% are indicated by the symbol *. Strains representing ex-type sequences are marked with “T’; ML, maximum likelihood; MP, maximum parsimony and the final alignment of 546 positions, including gaps. Differential patterns of ophiostomatoid fungal communities associated... S. polyporicola KX590768 T S inflata2 AY 495436 799 S. dimorphospora AY495439 T S. guttiliformis KX5S90778 T S. inflata AY495437T O. valdivianum KX590773 T S. dombeyi KX590767 T S. thermara KR051103 S. curviconia KX590777 T S. epigloea KX590760 T S. denticulatum KX590759 T S. candida HM041874 T S. aurorae DQ396800 T 5S. dimorphospora AY 495439 T S. stenoceras DQ296074 T S. stenoceras KX590756 S. eucastanea KX590753 T S. uta KU639616 S. globosa AM116966 T S. rossii KX590754T S. prolifera KX590770 T S. fusiformis AY 280461T -S. abietina KX590755T S. euskadiensis EF396344T S. gossypina KX590761T S. curviconia KX590776 0.05, Cc S. dimorphospora KX590806 T S. polyporicola KX590796 T S. cabrallii KX590804 T S. aemulophila KX590802 T S. candida KX590785 T S. chilensis KP711815 T |S. mexicana AM398393 T S. pallida KX590811T S. stylites KX590812T S. palmiculminata KX590794T S. bragantina KX590784 T S. brunneoviolacea KX590803 T S. fumea KX590788 T O. valdivianum KX590801 T og 9. Hebularis JQ438828 T S. nebularis JQ438829 T O. noisomeae KX590792 T S. prolifera KX590797 T S. lunata JQ511970T S. fusiformis JQ511967 T S. cantabriensis KF951540T S. gossypina JQ511972T S. curviconia JQ511968T S. euskadiensis JQ438830 T S. gossypina KX590789T S. abietina JQ511966 S. aurorae KX590783 T S. variecibatus KX590813 T S. eucastanea KX590781 T S. uat KU639605 S. stenoceras JQ511956 T q 0.04 109 Figure 9. Phylograms obtained from ML analysis of 8-tubulin A, C and calmodulin B, D sequences of Sporothrix, showing fungal associates with pines infected by Tomicus yunnanensis, I! minor and T. brevipilosus in Yunnan Province, China. Novel sequences obtained in this study are printed in bold type. Bootstrap values > 70% for ML and MP are indicated above branches. Bootstrap values < 70% are indicated by the symbol *, Strains representing ex-type sequences are marked with “T’; ML, maximum likelihood; MP, maximum parsimony and the final alignment of 284(A), 622(B), 260(C), 675(D) positions, including gaps. In the ITS-based phylogenetic analysis, strains of groups G and I were grouped with the O. minus complex (Fig. 6). ITS- and TUB2-based phylogenetic analyses consist- ently showed that group G strains formed a well-supported subclade between the North American and European subclades within the O. minus lineage (Fig. 7c, d). The strains of group G are therefore identified as O. minus. The ITS- and TUB2-based phylogenetic analyses consistently showed that the single strain of group I formed a branch that is related to, but distinct from the O. minus, O. kryptum and O. olgensis clades (Figs 6, 7d). Hence, this strain is interpreted as belonging to a distinct, undescribed Ophiostoma. 110 Hui Min Wang et al. / MycoKeys 50: 93-133 (2019) The remaining two groups (F and H) were not placed in any defined complex. Phylogenetic analyses, based on ITS and 7UB2 sequences, consistently showed that the group H strains clustered in the near vicinity of the O. tingens clade whereas group F strains formed a clade related to, but distinct from the O. macrosporum and. O. tin- gens clades (Figs 6, 7e). Thus, the strains in group H should be identified as O. tingens whereas the strains of group F represent an undescribed Ophiostoma. Strains of groups J and K nested within the Sporothrix lineage in LSU-based phy- logenetic analysis (Fig. 2). The phylograms resulting from the analyses of individuals are shown in Fig. 8 (ITS), Fig. 9a, c (TUB2) and Fig. 9b, d (CAL). The ITS-based analyses showed that group K strains belonged to the S. gossypina- complex whereas the group J strains were not placed in any species complex as defined by de Beer et al. (2016) (Fig. 8). Both groups formed independent, well-supported clades in ITS-, TUB2- and CAL-based phylogenetic analyses (Figs 8, 9). It could be deduced from results of multiple phylogenies that both groups represent novel species. Morphology and taxonomy From a morphological perspective, strains of groups D, E and G appeared, overall, concordant with the descriptions or our own observations of reference strains, namely of O. brevipilosi, O. canum and O. minus, respectively. However, although strains of groups A, C, and H are phylogenetically close to E. vermicola, L. yunnanense and O. tingens, respectively, justifying, for the time being, conspecificity, their phenotype devi- ated slightly from published descriptions and/or our own observation of type material. The description of these species is extended. Strains of groups B, E J and K revealed unique combinations of phenotypes, allowing morphological distinction from their closest phylogenetic relatives; consequently, they are described below as new species. The strain of the stand-alone group I also may represent an undescribed species; how- ever, we refrain from describing it for the time being, waiting for more material to become available. Taxonomy Esteya vermicola J.Y. Liou, J.Y. Shih & Tzean, Mycol. Res. 103(2): 243. 1999. MycoBank MB450702 Fig. 10 Description. Sexual form: unknown. Asexual form: Hyalorhinocladiella-like. Conidiophores mononematous, micronema- tous; conidiophorous cells solitary, integrated, flask-shaped, with an inflated base (3.6—) 4.6-6.1 (—7.1) um in diam., the fertile hyphoid part (9.1—) 12.2-19.0 (—22.5) x (1.4-) 1.9-3.1 (—4.7) um, often crooked due to successive conidial development; conidia Differential patterns of ophiostomatoid fungal communities associated... Thi Figure 10.A—H Morphological characters of Esteya vermicola A, B upper and reverse of cultures on 2% MEA 20 d after inoculation C=H conidiogenous cells with lunate conidia I-M the cuticle of a nematode attached by many lunate conidia. Scale bars: 20 um (I, K, L); 10 um (C-H, J, M). 1-celled, asymmetrically ellipsoidal in face view, concave, lunate in side view, with a layer of adhesive mucus on the concave surface, ending slightly apiculate, hyaline, smooth, (8.0—) 10-12 (—13.1) x (3.3—) 3.4-4.5 (—5.1) um, containing an ovoid en- dospore-like structure. 112 Hui Min Wang et al. / MycoKeys 50: 93-133 (2019) Culture characteristics. Colonies on 2% MEA in the dark reaching 31 mm in diam. in 8 days at 25 °C, growth rate up to 5 mm/day at the fastest; colony margin smooth. Mycelium compact, somewhat floccose in the margin, white at first, gradu- ally discolouring to greyish-green, eventually dark green. Optimal growth temperature 25 °C, growth at 5 °C and 35 °C. Known substrate and host. Galleries of Zomicus yunnanensis in Pinus yunnanensis. Known insect vector. Jomicus yunnanensis. Known distribution. Yunnan Province, China. Specimen examined. CHINA, Yunnan, Jomicus yunnanensis galleries in Pinus yunnanensis, Dec. 2016, HM Wang, CFCC 52625 = CXY 1893. Note. Esteya vermicola is known only from an asexual, Hyalorhinocladiella-like state producing lunate and bacilliform conidia (Liou et al. 1999, Kubatova et al. 2000, Wang et al. 2009, 2014) that we also observed in various strains of E. vermicola with a different origin (Taiwan, Korea, Czech Republic). Our strain was identified as E. vermicola based on phylogenetic inferences and morphological characters. However, our strain differed from previous descriptions (Liou et al. 1999) in having only lunate conidia in vitro. The size of the lunate conidia of our strains (mostly 10 - 12 x 3.4 - 4.5 um) was similar to that reported for E. vermicola, viz. 9.9-11.9 x 3.44.5 um vs 8.2-11.1 x 3.5-3.7 um (Taiwan, Liou et al. 1999), 9.3-12.4 x 3.0-3.2 um (Czech Republic, Kubatova et al. 2000), 7.7—12.1 x 3.0-3.8 um (Korea, Wang et al. 2009) or 8.7-11.9 x 3.0-3.6 um (Brazil, Wang et al. 2014). This is the first report of £. vermicola from continental China. The species was originally isolated from Japanese black pine infected by the pinewood nematode Bur- saphelenchus xylophilus, in Taiwan (Liou et al. 1999). Since then, its distribution range has been extended to Japan and Korea, Europe (Czech Republic, Italy) and both North (USA) and South America (Brazil) (Liou et al. 1999, Kubatova et al. 2000, Wang et al. 2009, 2014, Li et al. 2018). This species is associated with various vectors, including the pinewood nematode, Oxoplatypus quadridentatus and the bark beetle Scolytus intricatus. It was isolated also from wooden packaging material infested by Bursaphelenchus rainulfi. Graphilbum anningense H. Wang, Q. Lu & Z. Zhang, sp. n. MycoBank MB828884 Fig. 11 Etymology. ‘anningense’ (Latin), referring to the type locality. Type. CHINA, Yunnan, 7omicus yunnanensis galleries in Pinus yunnanensis, Apr. 2017, HM Wang, holotype CXY 1939, culture ex-holotype CFCC 52631 = CXY 1939. Description. Sexual form: unknown. Asexual forms: Pesotum-like and Hyalorhinocladiella-like. Pesotum-like conidiophores abundant on 2% MEA, macronematous, synnematous, (150—) 210-293 (—336) um long including conidiogenous apparatus, the base dark brown, slightly widened, (6.7—) Differential patterns of ophiostomatoid fungal communities associated... its) Figure | 1. Morphological characters of Graphilbum anningense sp. n. A, B Upper and reverse of cultures on 2% MEA 8 d after inoculation C, D, G conidiogenous cells of Pesotum-like macronematal asexual state and conidia E, F, H conidiogenous cells of Hyalorhinocladiella-like asexual state and conidia. Scale bars: 50 um (C); 20 um (D); 10 um (E=H). 7.9-18.8 (—29.0) um wide anchored in the media by brown rhizoid-like hyphae, the apex slightly enlarging, fan-shaped; conidiogenous cells hyaline, thin-walled, aseptate, (15.3—) 21.0-35.5 (42) x (0.7—) 1.1-1.9 (—2.3) pm; conidia 1-celled, clavate, ellip- soid to ovoid with truncate base and rounded apex, hyaline, smooth, (3.1—) 3.6-6.3 (-9.7) x (1.4-) 1.6-2.2 (-2.5) um. Hyalorhinocladiella-like: conidiogenous cells macronematous or semi-macronematous, mononematous, hyaline, simple or loosely branched, thin-walled, aseptate, (4.5—) 10.8- 29.0 (47) x (1.5—) 1.72.3 (2.6) um; conidia hyaline, clavate to ellipsoid, with obtuse ends, 1-celled, aseptate, smooth, (3.7—) 4.5—6.4 (—9.0) x (1.4—) 1.7-2.3 (-2.9) um. Culture characteristics. Colonies on 2% MEA in the dark reaching 90 mm in diam. in 6 days at 25 °C, growth rate up to 19.5 mm/day at the fastest; colony margin smooth. Mycelium superficial to flocculose or floccose, hyaline; reverse hyaline to pale yellowish. Optimal growth temperature 30 °C, slow growth at 40 °C, no growth at 5 °C. Known substrate and hosts. Galleries of Zomicus yunnanensis and T! minor in Pinus yunnanensis. Known insect vectors. Jomicus yunnanensis, I’ minor. Known distribution. Yunnan Province, China. Additional specimens examined. CHINA, Yunnan, Jomicus yunnanensis, T: mi- nor galleries in Pinus yunnanensis, Apr. 2017, HM Wang, CFCC 52632 = CXY 1940, CFCC 52633 = CXY 1944. 114 Hui Min Wang et al. / MycoKeys 50: 93-133 (2019) Note. Graphilbum anningense is characterised by a Pesotum-like and a Hyalorhi- nocladiella-like asexual state. It is phylogenetically closely related to Gra. rectangulo- sporium. However, Gra. rectangulosporium produced a sexual state in vitro (Ohtaka et al. 2006) which has not been observed in Gra. anningense. Other morphologically similar species include Gra. fragrans, Gra. crescericum, Gra. kesiyae and Gra. puerense. Graphilbum kesiyae and Gra. puerense also produce a Pesotum-like and a Hyalorhi- nocladiella-like asexual state. Graphilbum anningense and Gra. kesiyae differ by the size of their synnemata, whose length ranges do not overlap, viz. 210-293 um and 112.5-173 um long (Harrington et al. 2001), respectively. They also differ by their optimal growth temperature, respectively 30°C and 25°C. The synnemata of Gra. puerense, 206-357 um long (Chang et al. 2017), are marginally longer than those of Gra. anningense. Graphilbum fragrans and Gra. crescericum produce only a Lep- tographium-like and/or a Hyalorhinocladiella-like asexual state in vitro (Harrington et al. 2001, Chang et al. 2017). Graphilbum anningense was isolated from galleries of 7’ yunnanensis and T: minor infesting P yunnanensis. Previously, Gra. fragrans had been reported from 7) yunnan- ensis infesting P yunnanensis and from Pissodes spp. infesting Tsuga dumosa and P ar- mandii in China (Paciura et al. 2010, Zhou et al. 2013). Graphilbum kesiyae and Gra. puerense were isolated from galleries of Polygraphus aterrimus, Po. szemaoensis and Ips acuminatus infesting P kesiya (Chang et al. 2017). Although the geographic distribu- tion of these four Graphilbum species overlaps, their hosts and vectors are nevertheless, as far as it is known, different (Chang et al. 2017). Leptographium yunnanense X.D. Zhou, K. Jacobs, M.J. Wingf. & M. Morelet, Mycoscience 41(6): 576. 2000. MycoBank MB 466542 Fig. 12 Description. Sexual form: unknown. Asexual form: Leptographium-like. Conidiophores occurring singly or in groups of up to three, arising from the superficial mycelium, erect, macronematous, mon- onematous, (93.5—) 159-412 (—544) um long, without rhizoid-like structures; stipes simple, cylindrical, not constricted at septa, 1-6-septate, pale olivaceous at the base, (12—) 19.0-128 (—245) x (3.3—-) 4.1-6.1 (—7.3) um; conidiogenous apparatus (33.0-) 65.5-119.5 (—168.0) um long (high), with 2 to 3 series of cylindrical branches; pri- mary branches hyaline to pale olivaceous, smooth, cylindrical, 2-3 septate, (11.5-) 18.2-37.7 (—56.0) um long and (3.0—) 3.7—-5.9 (—7.7) um wide; secondary branch- es hyaline, 0-2 septate, (10.3—) 14.5-30.0 (—50.1) um long, (2.8—) 3.4-5.5 (-7.3) um wide; conidiogenous cells discrete, 2-3 per branch, cylindrical, (10.2—) 13.2-29.6 (—57.4) x (2.2—) 2.9-3.9 (4.4) um; conidia 1-celled, oblong to obovoid with truncate bases, hyaline, (5.8—) 7.0-10.4 (-13.0) x (2.9) 3.6-5.3 (-6.4) pm. Differential patterns of ophiostomatoid fungal communities associated... 115 Figure 12. Morphological characters of Leptographium yunnanense A, B upper and reverse of cultures on 2% MEA 8 d after inoculation D, I conidiophore on 2% MEA C, E=H conidiogenous cells of Leptographium-like asexual state and conidia. Scale bars: 50 um (D, 1); 10 um (C, E=H). Culture characteristics. Colonies on 2% MEA medium fast growing in the dark, reaching 76 mm in diam. in 8 days at 25 °C, growth rate up to 20 mm/day at the fast- est; colony margin smooth. Hyphae submerged in agar with aerial mycelium, greenish- olivaceous to olivaceous, smooth, straight; reverse hyphae umber-brown to dark oliva- ceous. Optimal growth temperature 25 °C, slow growth at 5 °C and 30 °C. Known substrate and hosts. Jomicus yunnanensis and its galleries in Pinus yun- nanensis, galleries of 7) brevipilosus in P kesiya. Known insect vectors. Zomicus brevipilosus, I! yunnanensis. Known distribution. Yunnan Province, China. Specimens examined. CHINA, Yunnan, adults of Yomicus yunnanensis and their galleries in Pinus yunnanensis, Tomicus brevipilosus galleries in P kesiya. Apr 20175 RUVe Wane mC ECE 252609) = Nats 97 IO RE Ces 26 20h = OXY a1900; CECE? 621s GAN 04, (CEES 52627 SG hyel908 y CRGC 5262 5823 MY 917. CECE 52624.= CX 1925. 116 Hui Min Wang et al. / MycoKeys 50: 93-133 (2019) Note. The sole reproductive structure formed on MEA in L. yunnanense is a Lep- tographium-like state. Our strains were identified as L. yunnanense, based on phyloge- netic evidence and secondarily, on morphological features. However, our strains slight- ly deviated from L. yunnanense in having longer conidiophores, mainly 159-412 um vs mostly 74-227 (—233) um (Zhou et al. 2000) or 80-240 pm (Yamaoka et al. 2008). Furthermore, our strains grew faster than reported for the species, 76 mm vs 44 mm in 8 days at 25 °C (Zhou et al. 2000). Although our strains were slightly genetically and morphologically divergent, we are of the opinion that they enter into the current L. yunnanense species concept (e.g. sensu Zhou et al. 2000). Yamaoka et al. (2008) showed the genetic diversity of L. yunnanense in Yunnan to be higher than in other places, that which is confirmed by the present study. Leptographium yunnanense was originally described from Yunnan Province with only an asexual state (Zhou et al. 2000). Subsequently, mating of strains from different origins (Thailand, China and Japan) yielded the sexual state, which is formed by neck- less ascocarps and cucullate ascospores (Yamaoka et al. 2008). Leptographium yunnanense was the third most abundant species associated with 7’ yunnanensis in our study. A few strains also were isolated from T’ brevipilosus infesting P kesiya and none from T! minor. Ophiostoma aggregatum H. Wang, Q. Lu & Z. Zhang, sp. n. MycoBank MB828885 Fig. 13 Etymology. ‘ageregatum (Latin), reflects to the conidiophores aggregated in clusters. Type. CHINA, Yunnan, from Tomicus minor galleries in Pinus yunnanensis, Dec. 2016, HM Wang, holotype CXY 1876, culture ex-holotype CFCC 52615 = CXY 1876. Description. Sexual form: unknown. Asexual form: Leptographium-like. Conidiophores macronematous, mononematous, gathered in groups up to 5, (28.5—) 34-45.5 (-52) um long; stipes cylindrical, 1-2 septate, not constricted at septa, umber-brown to dark olivaceous, (6.3—) 7.3-14.5 (—18) um long x (2.2—) 3.1-4.6 (—5.8) um wide. Conidiogenous apparatus (22—) 26.5— 31 (—34) um long, with 2—3 series of cylindrical branches; primary branches olivaceous, smooth, cylindrical all over, (5.9—) 7.2-13.5 (—20.5) x (3—) 3.3-4.2 (4.6) um; conid- iogenous cells discrete, 2-3 per branch, aseptate, cylindrical, hyaline to pale umber, (5.8) 7.2-12.1 (-18.5) x (2.1-) 2.8-4.0 (-4.7) um; conidia 1-celled, globose, elliptical with truncate bases, hyaline to pale umber, (4.0—) 4.8-5.9 (—6.3) x (3.1—) 4.0-5.0 (5.6) pm. Culture characteristics. Colonies on 2% MEA fast growing in the dark, reaching 90 mm in diam. in 8 days at 25 °C, growth rate up to 13 mm/day at the fastest; colony margin smooth. Hyphae submerged and aerial, umber-brown to dark olivaceous, floc- culose or floccose; reverse hyphae umber-brown to dark olivaceous. Optimal growth temperature 25 °C, able to grow at 5 °C and 30 °C. No growth at 35 °C. Known substrate and hosts. Galleries of Zomicus yunnanensis and T! minor in Pinus yunnanensis. Differential patterns of ophiostomatoid fungal communities associated... 7 Figure 13. Morphological characters of Ophiostoma aggregatum sp. n. A, B upper and reverse of cultures on 2% MEA 8 d after inoculation C conidiomata on 2% MEA (bar = 50 um) D=H conidiogenous cells of Leptographium-like asexual state and conidia. Scale bars: 20 um (C); 10 um (D-H). Known insect vectors. Jomicus minor, I! yunnanensis. Known distribution. Yunnan Province, China. Additional specimens examined. CHINA, Yunnan, from Tomicus yunnanensis and 7’ minor galleries in Pinus yunnanensis, Dec. 2016, Apr. 2017, HM Wang, CFCC 52616 = CXY 1875, CFCC 52617 = CXY 1874. Note. Ophiostoma aggregatum produced a single asexual, Leptographium-like state in vitro. This species is phylogenetically closely related to O. macrosporum, O. tingens, 118 Hui Min Wang et al. / MycoKeys 50: 93-133 (2019) O. floccosum, O. tapionis and O. piliferum in LSU-, ITS- and TUB2-based phyloge- netic inferences. Ophiostoma ageregatum and O. tingens are shown to be sympatric in Yunnan pine forest; both taxa were isolated from galleries and adults of 77 minor and TL. yunnanensis infesting P yunnanensis (Table 2). Ophiostoma tingens was also reported from T° minor infesting P yunnanensis in Yunnan (Pan et al. 2017). Ophiostoma aggregatum and O. tingens differ in their asexual state. Ophiostoma aggregatum only produces a Leptographium-like state. Inversely, the asexual states of O. tingens are variable. Our strains produced a Pesotum-like and a Sporothrix-like state whereas previously, Francke-Grosmann (1952) and de Beer et al. (2013b) reported a Hyalorhinocladiella- to Raffaelea-like state in European strains. The origin of this vari- ability and its importance for taxonomy is uncertain. Ophiostoma macrosporum, O. floccosum, O. tapionis and O. piliferum also differ from O. aggregatum by their asexual state. Ophiostoma macrosporum and O. floccosum produce a Pesotum-like asexual state, O. tapionis a Hyalorhinocladiella-like state and O. piliferum produces a Sporothrix-like state (Francke-Grosmann 1952, Upadhyay 1981, Yamaoka et al. 2004, Linnakoski et al. 2008). Ophiostoma macrosporum and O. tingens were both originally described in Tri- chosporium as T. tingens var. macrosporum and T! tingens (Lagerberg 1927, Francke- Grosmann 1952). Batra (1967) transferred these two species into Ambrosiella. It is only recently that the morphological characteristics were found to agree with those of Ophiostoma (de Beer et al. 2013b). Ophiostoma macrosporum has been reported from various Pinus spp. (including P sylvestris) infected by [ps acuminatus in Europe (Francke-Grosmann 1952, Batra 1967). Ophiostoma tingens (Lagerb. & Melin) Z.W. de Beer & M.J. Wingf., Svensk Skogs- vardsférening Tidskr. 25:233. 1927. MycoBank: MB801091 Fig. 14 Description. Sexual form: unknown. Asexual forms: Pesotum-like and Sporothrix-like. Pesotum-like: conidiophores ma- cronematous, synnematous; synnemata simple, anchored into the substrate by brown rhizoid-like hyphae, (333-—) 344-584 (—684) um long including conidiogenous appara- tus, the base dark brown, slightly widened, (16.7—) 17—50.5 (—65.5) um wide, the apex cream-coloured or pale brown, slightly widening; conidia hyaline, globose to elliptical, 1-celled, smooth, (2.7—) 3.6—7.2 (—8.0) x (2.8-) 4.3-6.1 (-7.0) um. Sporothrix-like: conidiophores semi-macronematous, mononematous, hyaline, sim- ple or loosely branched, smooth, bearing terminal denticulate conidiogenous cells (8.3—) 15.6-30.0 (42.5) x (1.1-) 1.7—3.1 (-4.7) um; conidia hyaline, globose to ellipti- cal, obovoid with pointed bases and rounded apices, 1-celled, smooth, (2.6—) 4.0-6.8 (-8.7) x (2.2—) 3.5—-5.5 (—7.4) um. Differential patterns of ophiostomatoid fungal communities associated... 119 Figure | 4. Morphological characters of Ophiostoma tingens A,B upper and reverse of cultures on 2% MEA 20 d after inoculation C=G conidiogenous cells of Sporothrix-like asexual state and conidia H-J conidiogenous cells of Pesotum-like macronematal asexual state and conidia. Scale bars: 10 pm (C=-H); 50 um (I, J). Culture characteristics. Colonies on 2% MEA medium slow growing in the dark, reaching 39 mm in diam. in 8 days at 25 °C, growth rate up to 5 mm/day at the fastest; colony margin anomalous. Hyphae appressed to flocculose, black; reverse hyphae also black. Optimal growth temperature 25 °C, no growth at 5 °C and 30 °C. Known substrate and hosts. Galleries of Zomicus yunnanensis and T! minor in Pinus yunnanensis. Known insect vectors. Jomicus yunnanensis, T’ minor. Known distribution. Yunnan Province, China; Europe. Specimens examined. CHINA, Yunnan, from Yomicus minor and T! yunnanensis galleries in Pinus yunnanensis, Feb. 2017, Nov. 2016, HM Wang, CFCC 52611 = CXY 1866, CFCC 52612 = CXY 1865, CFCC 52613 = CXY 1868. Note. Our strains of O. tingens were identified based on phylogenetic affinities and morphological features. (cf. above under note for O. aggregatum.) 120 Hui Min Wang et al. / MycoKeys 50: 93-133 (2019) Ophiostoma tingens has been reported from sapwood of various Pinus spp. (including P sylvestris) infested by 7’ minor, T; piniperda and Ips sexdentatus in Europe (Francke-Gros- mann 1952, Batra 1967, Jankowiak 2008). The species was recorded in Yunnan Province in China in 2017, associated with 7’ minor infesting P yunnanensis (Pan et al. 2017). Sporothrix macroconidia H. Wang, Q. Lu & Z. Zhang, sp. n. MycoBank: MB828886 Fig. 15 Etymology. ‘macroconidia (Latin), referring to the large conidia of this fungus. Type. CHINA, Yunnan, from Yomicus yunnanensis galleries in Pinus yunnanensis, Dec. 2016, collected by HM Wang, holotype CXY 1894, culture ex-holotype CFCC 52628 = CXY 1894. Description. Sexual form: unknown. Asexual form: Sporothrix-like. Conidiophores semi-macronematous, mononema- tous; conidiogenous cells hyaline, simple or loosely branched, thin-walled, aseptate, bear- ing denticles forming a rachis (4.1—) 11.0-24.5 (—36.5) x (1.4) 2.1-3.4 (-4.9) um; conidia hyaline, cylindrical, ellipsoid to ovoid, 1-celled, smooth, (3.6—) 4.8—7.4 (—9.9) x (2.5—) 3.2-4.9 (—9.9) um, solitarily or aggregating in slimy masses. Culture characteristics. Colonies on 2% MEA medium slow growing in the dark, reaching 34 mm in diam. in 8 days at 25 °C, growth rate up to 5 mm/day at the fastest; colony margin smooth. Hyphae appressed to flocculose, white; reverse hyaline to pale yellowish. Optimal growth temperature 25 °C, little growth at 5 °C and 35 °C. Known substrates and hosts. Galleries of Tomicus yunnanensis and T: brevipilosus in Pinus yunnanensis and P kesiya. Known insect vectors. Zomicus yunnanensis, 1; brevipilosus. Known distribution. Yunnan Province, China. Additional specimens examined. CHINA, Yunnan, from Tomicus brevipilosus galleries in Pinus kesiya, Dec. 2016, Jan. 2017, HM Wang, CFCC 52629 = CXY 1895, CFCC 52630 = CXY 1896. Note. Sporothrix macroconidia is closely related to O. valdivianum, S. bragan- tina, S. brunneoviolacea and S. fumea in phylogenetic analyses inferred from LSU, ITS, TUB2 and CAL DNA sequence data. It differs from these species by its co- nidia, which are larger than those of the other four species, mostly 4.8—7.4 x 3.2-4.9 um and 4-6 x 2 um in O. valdivianum (Butin and Aquilar 1984), 4—6 x 2-2.5 um in S. bragantina (Pfenning and Oberwinkler 1993), 3-7 x 1.5-3 um in S. brunneoviolacea (Madrid et al. 2010) and 1.5—2.0 x 0.5-1.0 um in S. fumea (Nkuekam et al. 2012). In addition, a sexual state was observed in vitro for O. val- divianum, S. bragantina and S. fumea, which was not observed in S. macroconidia and S. brunneoviolacea. Sporothrix macroconidia was found associated with 7! yunnanensis infesting P yun- nanensis and with 1. brevipilosus infesting P kesiya. The other four similar species have Differential patterns of ophiostomatoid fungal communities associated... 1A Figure 15. Morphological characters of Sporothrix macroconidia sp. n. A, B Upper and reverse of cultures on 2% MEA 20 d after inoculation C-H conidiogenous cells of Sporothrix-like asexual state and conidia. Scale bars: 10 um (C=H). very different ecology and known geographic distributions. Sporothrix fumea was iso- lated from Eucalyptus cloeziana infested by Phoracantha beetles in South Africa (Nkue- kam et al. 2012), whereas O. valdivianum, S. bragantina and S. brunneoviolacea were obtained from soil or Nothofagus in Europe and South America (Butin and Aquilar 1984, Pfenning 1993, Madrid et al. 2010). Sporothrix pseudoabietina H. Wang, Q. Lu & Z. Zhang, sp. n. MycoBank: MB828887 Fig. 16 Etymology. ‘pseudoabietina (Latin), referring to the phylogenetic affinities to S. abietina. Type. CHINA, Yunnan, from 7. minor galleries in P yunnanensis, Apr. 2017, HM Wang, holotype CXY 1937, culture ex-holotype CFCC 52626 = CXY 1937. Description. Sexual form perithecial: on 2% MEA, perithecia superficial or par- tially immersed, with a globose base extending into a cylindrical neck, often termi- nated by ostiolar hyphae; bases (85—) 110-152 (—168) um diam., black, the outer layer with dark brown hyphal ornamentation; apical neck mild to dark brown at the 122 Hui Min Wang et al. / MycoKeys 50: 93-133 (2019) Figure 16. Morphological characters of Sporothrix pseudoabietina sp. n. A, B upper and reverse of cul- tures on 2% MEA 20 d after inoculation C, D ostiolar hyphae present E, F perithecium G ascospores of sexual state H=I conidiogenous cells of Sporothrix-like asexual state and conidia. Scale bars: 20 um (C, D); 50 um (E, F); 10 um (G-I). base, pale brown to pale yellow or hyaline toward the apex, straight or slightly curved, (172—) 560-985 (—1039) um long, (37—) 41-62 (—78) wm wide at the base, (9.3-) 12.5-17.5 (—20) wm wide at the apex; ostiolar hyphae numerous, hyaline, divergent, (19.5—) 21.5-38.0 (-43) um long; asci not seen; ascospores hyaline, 1-celled, orange- shaped in lateral view, ellipsoid in face view, circular in polar view, (2.9 —) 3.4-4.4 (—5.3) x (0.8—) 1.0-1.5 (—1.9) um, without mucilaginous sheath. Asexual form: Sporothrix-like. Conidiophores semi-macronematous to mononema- tous; conidiogenous cells hyaline, simple or loosely branched, smooth, bearing denticles disposed in a dense rachis (16.0—) 20.5—30.5 (—34.5) x (1.2—) 1.6-2.0 (—2.3) um; conidia \-celled, clavate, ellipsoid to ovoid, hyaline, (3.0—) 4.0-7.0 (-9.0) x (1.0-) 1.1-3.1 (4.8) pm. Culture characteristics. Colonies on 2% MEA slow growing in the dark, reaching 23 mm in diam. in 8 days at 25 °C, growth rate up to 2.5 mm/day at the fastest; colony margin smooth. Hyphae appressed to flocculose or floccose, white; reverse hyaline to pale yellowish. Optimal growth temperature 25 °C; very slow growth at 35 °C; no growth at 5 °C. Known substrate and hosts. Galleries of Zomicus yunnanensis and T! minor in Pinus yunnanensis. Known insect vectors. Jomicus yunnanensis, I! minor. Differential patterns of ophiostomatoid fungal communities associated... 125 Known distribution. Yunnan Province, China. Additional specimen examined. CHINA, Yunnan, Jomicus minor galleries in Pi- nus yunnanensis, Apr. 2017, HM Wang, CFCC 52627 = CXY 1938. Note. Sporothrix pseudoabietina is characterised by a perithecial sexual form and a Sporothrix-like asexual state. Multiple phylogenetic inferences (LSU, ITS, TUB2 and CAL) showed that S. pseudoabietina belonged to the S. gossypina complex, in which it is closely related to S. abietina. However, it can be distinguished from this species, based on both morphological and physiological features. The conidia of S. pseudoabietina (4.0-7.0 x 1.1-3.1 um) are wider than those of S. abietina (4—7.5 x 1-2 um) (Mar- molejo and Butin 1990). Perithecia are known from S. abietina but only on natural substrates and not in vitro on artificial media, contrary to those from S. pseudoabietina. The perithecial neck in S. pseudoabietina is much longer than that of S. abietina, viz. mostly 560-985 um and 450-650 um, respectively. Ostiolar hyphae of S. abietina and S. pseudoabietina also differ in number, numerous vs 7—10 and size, mostly 13-19 um and in S. pseudoabietina 21.5-38.0 um (Fig. 11c, d). In addition, no growth of S. abi- etina was observed at 35 °C, but S. pseudoabietina can grow at 35 °C. The hosts and geographic distributions of S. pseudoabietina and S. abietina are also very different. Sporothrix pseudoabietina was found associated with 77 minor and T; yunnanensis infecting P yunnanensis, whereas S. abietina was reported from Abies vejari attacked by Pseudohylesinus sp. in Mexico (Marmolejo and Butin 1990). Discussion In this study, 772 strains of ophiostomatoid fungi were isolated from galleries and adults of three pine shoot beetles, 77 brevipilosus, T’ minor and T; yunnanensis, inhabit- ing P yunnanensis and P. kesiya in forests in Yunnan Province, south-western China. Multiple phylogenetic analyses and morphological features allowed the identification of 11 species from 5 genera. Six species corresponded to known taxa (E. vermicola, L. yunnanense, O. brevipilosi, O. canum, O. minus and O. tingens), whereas four species are proposed as new, Gra. anningense, O. aggregatum, S. pseudoabietina and S. macroco- nidia. A single strain remained unnamed. The global ophiostomatoid fungal communities, associated with these three Tomi- cus species in pine forest, were dominated by far by three species, which are, in decreas- ing order of isolates, O. canum, O. brevipilosi and O. minus. Furthermore, these three ophiostomatoid species are not equally associated with the three Zomicus species but show variable degrees of preference or specificity. Overall, O. canum was the most frequently isolated species in our study (253 out of the 772 strains). It was preferably (79.4% of the O. canum strains) isolated from gal- leries and adults of 7’ minor, infesting both P yunnanensis and P kesiya (Table 3) and dominated the ophiostomatoid community associated with this beetle (81.4%, 201 strains of O. canum out of 247 strains in the community, Table 3). This is the first report of this species in China. It was previously reported in eastern Asia but only in Japan (Masuya et al. 1999). Ophiostoma canum was also shown to be 124 Hui Min Wang et al. / MycoKeys 50: 93-133 (2019) the dominant species associated with 7’ minor, both in Europe and Japan (Masuya et al. 1999, Jankowiak 2008). In addition, this species was found in association with other bark beetles in Finland and Russia, e.g. Hylastes brunneus, Hylurgops palliatus, Ips ty- pographus, Pityogenes chalcographus and Trypodendron lineatum (Linnakoski et al. 2010). The close association between O. canum and T. minor appears stable over an extensive geographical distribution and tree host range, indicating likely intimate relationships. Ophiostoma brevipilosi represented the second most frequently isolated species in our survey (224 out of 772 strains), occurring exclusively in galleries and adults of 7 brevipilosus, dominating this beetle’s ophiostomatoid community (98.2%, 224 strains of O. brevipilosi out of 228 strains in the community, Table 3). The occurrence or fit- ness of O. brevipilosi is therefore strongly linked to the presence of 7’ brevipilosus. Ophiostoma brevipilosi was described originally from Yunnan, based on six strains, all isolated from 7 brevipilosus (Chang et al. 2017). It belongs to the recently defined O. clavatum complex (Linnakoski et al. 2016). It is only known from this area of south-western China. Ophiostoma minus was the third most frequently isolated species overall (197 strains out of 772), occurring exclusively in galleries and adults of 7’ yunnanensis infesting P yunnanensis, dominating this beetle ophiostomatoid community (66.3%, 197 strains of O. minus out of 297 strains in the community, Table 3). Ophiostoma minus, first reported as a blue-stain agent in Europe (Munch 1907), is a widely distributed species, also recorded from North America and East Asia (Japan and China) (Hedgcock 1906, Gorton and Webber 2000, Gorton et al. 2004, Lu et al. 2009, Linnakoski et al. 2010). It infests various pines and is transported by various bark beetles. This species was predominantly associated with 7) piniperda in Europe (Jankowiak 2006) and Japan (Masuya et al. 1999) and with the southern pine beetle, Dendroctonus frontalis, in the southern states of the USA (Klepzig 1998, Gorton and Webber 2000, Gorton et al. 2004). Ophiostoma minus was deemed to have two allopatric populations, viz. a North American and a Eurasian population (Gorton et al. 2004). In ITS/ TUB2 phylogenetic inferences, the North American and Eurasian populations of O. minus were resolved as two closely related clades (Gorton et al. 2004, Lu et al. 2009). ITS and 7UB2-based phylogenetic inferences (Fig. 7c, d) also resolved our strains as a third distinct clade, which could thus be interpreted as a third allopatric population. The question of trans- lating these populations into a Linnaean taxonomic rank, however, remains open. Tomicus yunnanensis galleries and adult beetles harboured the highest diversity of ophiostomatoid fungi; ten of the 11 species identified were isolated from galleries and adults of this beetle. Three species were exclusively found with this beetle (O. minus, E. vermicola, Ophiostoma sp. 1). By comparison, galleries and adults of 7 minor and of T: brevipilosus yielded less species; five species were isolated from T’ mi- nor, none of which was associated exclusively with this beetle and three species from LT. brevipilosus, of which one was exclusive, O. brevipilosi. Five species are shared by both 7? yunnanensis and T’ minor and two species by both 7! yunnanensis and T: brevipilosus, but none by T! minor and T! brevipilosus and also none by all three pine shoot beetles (Table 3, Fig. 17). Differential patterns of ophiostomatoid fungal communities associated... 125 T. yunnanensis T. brevipilosus Figure 17. Venn diagram showing overlaps of the ophiostomatoid fungal communities associated with three pine shoot beetles. The ectosymbiosis between bark beetles and fungi is widespread and diverse. Some fungi are highly specific and associated with a single beetle species, forming a ‘species-specific association’ (Six and Paine 1999, Six 2012), while others can be associated with many vectors (Kostovcik et al. 2014). The species-specific associations include, for instance, [ps typographus and Endoconidiophora polonica, I. cembrae and End. laricicola (Harrington et al. 2002) or 1. subelongatus and End. fujiensis (Marin et al. 2005, Meng et al. 2015). The present study showed that species-specific associations might occur with various sympatric beetles that share the same niche. The association of 7 brevipilosus and O. brevipilosi seems to be species-specific in the pine forest of Yunnan, where both taxa are, so far, endemic. In the pine forest of Yunnan, the Chinese ‘population’ of O. minus is also specifically associated with I) yunnanensis, whereas the two other O. minus ‘populations’ are associated, at least preferably, with Dendroctonus frontalis and T. piniperda (Gorton et al. 2004, Jankowiak 2006). The genetically distinct ‘populations’ might originate from both 126 Hui Min Wang et al. / MycoKeys 50: 93-133 (2019) the allopatric distribution and vector specificity and both factors could support recognition of three distinct taxa. In the pine forest of Yunnan, the association of O. canum with T- minor is preferential but not exclusive. Up to now, no data have been provided proving the pathogenicity of these ophi- ostomatoid species to both indigenous pines, except for L. yunnanense (Liao and Ye 2004, Gao et al. 2017). Pathogenicity tests have been done by artificial inoculation of the dominant species into seedlings of the two pines. The results preliminarily showed that the virulence of O. minus and O. brevipilosi was significantly stronger than that of O. canum. This is similar to the relative aggressive nature of the three 7omicus spe- cies. Thus, we suspect there might be some link between beetle aggression and fungus virulence (Christiansen et al. 1987, Kirisits 2004). Conclusions This study provides evidence for the diversity of ophiostomatoid fungi associated with TL. yunnanensis, 1: minor and T: brevipilosus in Yunnan pine forest in south-western China. Eleven species were identified, of which four were new to science. The diversity is the highest in the galleries and adults of 7’ yuwnnanensis and the poorest in the galler- ies and adults of 7’ brevipilosus. Three species, namely O. brevipilosi, O. canum and O. minus, dominate the ophi- ostomatoid communities; each is associated predominantly with one species of Tomi- cus, namely T! brevipilosus, I! minor and T. yunnanensis, respectively. In this regard, this study has revealed differential associations between beetles living sympatrically, concomitantly or sequentially, in the same ecological niche, which indicates a certain level of specificity of the relationships between the fungi and the beetles. However, the parameters behind these (partial) species-specific relationships remain unknown. Increased study of the biodiversity, biogeography and ecology of ophiostomatoid fungi in China, in particular of those associated with Tomicus spp., would facilitate comparison with well-known species associated with other Zomicus spp. in other neigh- bouring or distant geographical areas, e.g. in European countries, Japan and Korea and allow a better understanding of the occurrence and mechanisms behind the outbreak of infections, enabling the development of effective management methods to alleviate the subsequent plant losses. Acknowledgements This study was supported by the National Natural Science Foundation of China (Pro- ject No.: 31770693 and 31770682). We are very grateful to Shuangcheng Li and Hongxun Wang for their help in field survey and collection. Cony Decock gratefully acknowledges the financial support received from the Belgian State—Belgian Federal Science Policy through the BCCM programme. Differential patterns of ophiostomatoid fungal communities associated... 127 References Batra LR (1967) Ambrosia fungi: a taxonomic revision, and nutritional studies of some species. Mycologia 59: 976-1017. https://doi.org/10.2307/3757271 Ben Jamaa ML, Lieutier EF, Yart A, et al. (2007) The virulence of phytopathogenic fungi associ- ated with the bark beetles Zomicus piniperda and Orthotomicus erosus in Tunisia. For. 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