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

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 Tomicus spp. (T.brevipilosus, T.minor and T.yunnanensis) infect branches and trunks of Pinusyunnanensis and P.kesiya in Yunnan Province, in south-western China. Tomicus spp. are well known as vectors of ophiostomatoid fungi and their co-occurrence could result in serious ecological and economic impact on local forest ecosystems. 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 Tomicus 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, including 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 β-tubulin (TUB2), elongation factor 1α (TEF1-α) and calmodulin (CAL). Phylogenetic analyses were performed using maximum parsimony (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, Esteyavermicola, Leptographiumyunnanense, Ophiostomabrevipilosi, O.canum, O.minus and O.tingens and four novel taxa, described as Graphilbumanningense, O.aggregatum, Sporothrixpseudoabietina 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, although harbouring a diversity of ophiostomatoid species, was differentially dominated by a single fungal species; Ophiostomacanum was preferentially associated with and dominated the ophiostomatoid community of T.minor, whereas O.brevipilosi and O.minus were exclusively associated with and dominated the ophiostomatoid communities of T.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 Tomicus populations are dominated by distinct species showing some level of specificity or even exclusivity.


Introduction
Associations between insects and microorganisms are increasingly recognised as one of the major issues in forest ecology and forest health around the world . Many bark beetles are well known as tree pests causing various levels of tree mortality and forest decline in large areas of the world, mostly in temperate areas (Jankowiak 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).
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, Lu et al. 2014. Although T. brevipilosus is able to infect healthy trees, it preferably colonises trunks already infested by T. yunnanensis or both T. yunnanensis and T. minor (Chen et al. 2010(Chen et al. , 2015. Tomicus minor is often regarded as a secondary, opportunist species infesting trees already weakened by T. yunnanensis or/and T. brevipilosus (Ye and Ding 1999, Lieutier et al. 2003, Chen et al. 2009).
Pine shoot beetles such as T. piniperda, T. minor and T. destruens are commonly associated with ophiostomatoid fungi (Masuya et al. 1999, Kim et al. 2005, Jankowiak 2006, 2008. Fifteen ophiostomatoid fungi were reported associated with T. piniperda in Europe (Mathiesen 1950, Lieutier et al. 1989, Gibbs and Inman 1991, Solheim and Långström 1991, Jankowiak 2006, Jankowiak and Bilański 2007 and 11 were documented in eastern Asia (Japan and Korea) (Masuya et al. 1999, Kim et al. 2005. Ophiostoma minus was shown to be the dominant species associated with T. 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 pathogenic one (Gibbs and Inman 1991) in Europe. Tomicus minor also infests various pines in Europe and Asia. Fifteen (Mathiesen-Käärik 1953, Masuya et al. 1999, Jankowiak 2008 and 11 (Masuya et al. 1999) ophiostomatoid species have been reported to be associated with this beetle species in Europe and Japan, respectively. Ophiostoma canum was recorded as a frequent/dominant species in association with T. 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 T. destruens in Europe (Lieutier 2002, Sabbatini Peverieri et al. 2006, Ben Jamaa et al. 2007. Despite the fact that Tomicus spp. have caused serious losses to forest ecosystems in south-western China, there are no systematic studies of their ophiostomatoid associates but only a few sporadic reports. So far, nine ophiostomatoid species have been reported as being associated with Tomicus spp. in Yunnan. Six species (Leptographium yunnanense, Ophiostoma ips, O. minus, O. quercus, S. abietina and S. nebularis) were recorded to be associated with T. yunnanensis (Ye et al. 2000, Chang et al. 2017. Two species (Graphilbum fragrans and O. tingens) were recorded as being associated with T. minor (Zhou et al. 2013, Pan et al. 2017, whereas only a single species (O. brevipilosi) was recorded as being associated with T. brevipilosus (Chang et al. 2017). Amongst them, L. yunnanense was the first species newly described from the area  and is likely the most virulent one (Liao andYe 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.
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 (Christiansen et al. 1987, Kirisits 2004, Linnakoski et al. 2012, although this has been questioned 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 various beetle species co-exist.
The aim of this study was to describe the diversity of ophiostomatoid fungal communities associated with three pine shoot beetles and their galleries infesting P. yunnanensis 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 outbreaks and rapid expansion of the pests.

Sample collection and fungus isolation
Samples of galleries in bark and shoots and adults of Tomicus spp. were collected from P. yunnanensis and P. kesiya at five sites in Yunnan Province (Fig. 1, Table 1) from December 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 processed 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 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 cultures were made to observe all microscopic characters (sexual/asexual structures) using a BX51 OLYMPUS microscope with differential interference contrast. Fifty measurements were made of each relevant structure and the ranges were calculated. Standard 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. Sequences missing data are indicated by [-] deviation (SD), minimum (min) and maximum (max) measurements are presented as (min-) (mean-SD) -(mean+SD) (-max). The optimal growth temperature of the various strains was determined by placing a 5-mm (diam.) plug from an actively growing fungal colony upside down at the centre of an MEA plate. For each strain, three replicates were incubated at temperatures ranging from 5 to 35 °C at five-degree intervals, for 8d. The diameter of each colony was measured daily. Culture characters were recorded on MEA incubated at 25 °C for 8 d and 20 d. Colour descriptions were made by reference to Rayner (1970).

DNA extraction and sequencing
DNA was extracted from actively growing mycelium scraped from seven-day-old cultures using sterile scalpels and transferred to 2 ml Eppendorf tubes. DNA extraction and purification were performed using the Invisorb Spin Plant Mini Kit (Invitek, Berlin, Germany), following the manufacturer's protocols.

Phylogenetic analyses
BLAST searches for the obtained sequences were performed in NCBI GenBank and published sequences of closely related species were downloaded. Alignments of the genes were made using MAFFT 7.0 (Katoh and Standley 2013) and the E-INS-i strategy and edited manually in MEGA 5.2 (Tamura et al. 2011). Phylogenetic analyses were performed using maximum parsimony (MP) as well as maximum likelihood (ML).
ML analyses were implemented using RAxML v. 7.0.3 (Stamatakis 2006), under the GTR-GAMMA model. Support for the nodes was estimated from 1 000 bootstrap replicates. The results were subsequently exported to Figtree v.1.4.2 to visualise the trees.
MP analyses were implemented in PAUP* 4.0b10 (Swofford 2003). The most parsimonious trees were identified by a heuristic search of 1 000 random addition sequence replicates, using the tree-bisection-recognition (TBR) algorithm for branch swapping. Branch support was assessed by 1 000 bootstrap replicates. Tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were used to evaluate the trees.

Fungal isolation and sequence comparisons
Three Tomicus species occurred on P. yunnanensis and P. kesiya in the areas studied, either independently or concomitantly in individuals of the host trees ( Fig. 1). In total, 772 strains of ophiostomatoid fungi (Hyalorhinocladiella-like, Ophiostoma, Pesotumlike, Leptographium-like and Sporothrix-like) were isolated from 223 adult beetles (20% of the strains) and 890 galleries (80% of the strains). Galleries or adults of T. yunnanensis yielded 297 strains whereas 247 strains were retrieved from galleries or adults of T. minor and 228 strains from galleries or adults of T. brevipilosus (Table 3).
The LSU sequence was used to search for preliminary affinities using the BLASTn search option in GenBank. As a result, these strains were found to be distributed over 5 genera and 11 tentative species/groups (A-K) ( Table 2).

Phylogenetic analyses
The degrees of polymorphism of LSU, ITS, TUB2, TEF1-α and CAL make them variably suitable for genus or species discrimination amongst ophiostomatoid fungi. The LSU sequence is a suitable marker to infer the generic affinities (de Beer and Wingfield 2013, de Beer et al. 2013aBeer et al. , 2016; it allowed confirming the preliminary placement of our strains based on morphological characters (Fig. 2). The ITS region would be useful to place strains within the Ophiostoma s. l. complex, but the degree of polymorphism does not allow distinguishing species. Usually, TUB2, TEF1-α and CAL regions are better markers to identify and, where appropriate, to show the genetic diversity within ophiostomatoid fungi (Zipfel et al. 2006, de Beer et al. 2016).
On the basis of the LSU blast searches, one to six strains of each tentative species (A-K) were selected for sequencing of five additional DNA markers (ITS, ITS2-LSU, TUB2, TEF1-α and CAL) to infer more accurate identification and phylogenetic affinities. Six sequence datasets (LSU, ITS, ITS2-LSU, TUB2, TEF1-α and CAL) were generated for a total of 31 representative strains (Table 2) and the sequences were deposited in GenBank. Resulting alignments were deposited in TreeBASE (submission no: 24032). The topologies generated by the ML and MP analyses were highly concordant and the ML phylograms are presented for all the individual genes, incorporating nodal supports of both the ML and MP analyses.
The LSU dataset consisted of 109 sequences, 11 sequences obtained in this study and 98 downloaded from GenBank. The phylogenetic analyses confirmed the morphology-based placement of our strains into Esteya, Graphilbum, Leptographium, Ophiostoma and Sporothrix (Fig. 2).
Group A consisted of a single strain. LSU-based phylogenetic analysis showed this strain to be close to E. vermicola (Fig. 2). TUB2 and TEF1-α data analysis confirmed the strain's close affinities to E. vermicola (Fig. 3a, b), that could justify conspecificity.
Group B strains nested within the Graphilbum lineage in the LSU-based phylogenetic analysis (Fig. 2). Phylogenetic analysis based on LSU, ITS and TUB2 concordantly showed that the group B strains formed a single, well-supported clade related to but distinct from Gra. rectangulosporium and Gra. microcarpum (Fig. 4a, b); this would warrant its recognition as a distinct, undescribed species.
Group C strains were shown to belong to the Leptographium lineage in the LSUbased phylogenetic analysis (Fig. 2). The ITS2-LSU dataset consisted of six of our own sequences and 49 reference sequences downloaded from GenBank. Within the Leptographium lineage, group C strains nested in the L. lundbergii-complex; they were related to L. yunnanense, L. lundbergii and L. conjunctum (Fig. 5a). TUB2-and TEF1-α based analysis confirmed their close affinities with L. yunnanense, although forming a slightly divergent clade (Fig. 5b, c). TUB2 and TEF1-α sequences of group C strains showed some polymorphisms, which could be considered as falling within the natural diversity of L. yunnanense.   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 lineages 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 retrieved from GenBank, representing the major groups of Ophiostoma (de Beer and , Linnakoski et al. 2016. The ITS-and TUB2-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 , 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.    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 consistently 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. The remaining two groups (F and H) were not placed in any defined complex. Phylogenetic analyses, based on ITS and TUB2 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. tingens 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 phylogenetic 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. gossypinacomplex 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 deviated slightly from published descriptions and/or our own observation of type material. The description of these species is extended. Strains of groups B, F, 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; however, we refrain from describing it for the time being, waiting for more material to become available. Asexual form: Hyalorhinocladiella-like. Conidiophores mononematous, micronematous; conidiophorous cells solitary, integrated, flask-shaped, with an inflated base (3.6-) 4.6-6.1 (-7.1) μm in diam., the fertile hyphoid part (9.1-) 12.2-19.0 (-22.5) × (1.4-) 1.9-3.1 (-4.7) μm, often crooked due to successive conidial development; conidia 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) × (3.3-) 3.4-4.5 (-5.1) μm, containing an ovoid endospore-like structure.  Note. Esteya vermicola is known only from an asexual, Hyalorhinocladiella-like state producing lunate and bacilliform conidia (Liou et al. 1999, Kubátová et al. 2000, Wang et al. 2009) 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 × 3.4 -4.5 μm) was similar to that reported for E. vermicola, viz. 9.9-11.9 × 3.4-4.5 μm vs 8.2-11.1 × 3.5-3.7 μm (Taiwan, Liou et al. 1999), 9.3-12.4 × 3.0-3.2 μm (Czech Republic, Kubátová et al. 2000), 7.7-12.1 × 3.0-3.8 μm (Korea, Wang et al. 2009) or 8.7-11.9 × 3.0-3.6 μm (Brazil, Wang et al. 2014). This is the first report of E. vermicola from continental China. The species was originally isolated from Japanese black pine infected by the pinewood nematode Bursaphelenchus 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, Kubátová et al. 2000, Wang et al. 2009, 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. Fig. 11 Etymology. 'anningense' (Latin), referring to the type locality.
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  Note. Graphilbum anningense is characterised by a Pesotum-like and a Hyalorhinocladiella-like asexual state. It is phylogenetically closely related to Gra. rectangulosporium. 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 Hyalorhinocladiella-like asexual state. Graphilbum anningense and Gra. kesiyae differ by the size of their synnemata, whose length ranges do not overlap, viz. 210-293 μm and 112.5-173 μm 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 μm long (Chang et al. 2017), are marginally longer than those of Gra. anningense. Graphilbum fragrans and Gra. crescericum produce only a Leptographium-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 T. yunnanensis and T. minor infesting P. yunnanensis. Previously, Gra. fragrans had been reported from T. yunnanensis infesting P. yunnanensis and from Pissodes spp. infesting Tsuga dumosa and P. armandii 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 distribution of these four Graphilbum species overlaps, their hosts and vectors are nevertheless, as far as it is known, different (Chang et al. 2017). Description. Sexual form: unknown.

Known substrate and hosts. Tomicus yunnanensis and its galleries in
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 . Subsequently, mating of strains from different origins (Thailand, China and Japan) yielded the sexual state, which is formed by neckless ascocarps and cucullate ascospores (Yamaoka et al. 2008).
Leptographium yunnanense was the third most abundant species associated with T. yunnanensis in our study. A few strains also were isolated from T. brevipilosus infesting P. kesiya and none from T. minor. Fig. 13 Etymology. 'aggregatum' (Latin), reflects to the conidiophores aggregated in clusters.
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, flocculose 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 Tomicus yunnanensis and T. minor in Pinus yunnanensis. O. floccosum, O. tapionis and O. piliferum in LSU-, ITS-and TUB2-based phylogenetic inferences. Ophiostoma aggregatum and O. tingens are shown to be sympatric in Yunnan pine forest; both taxa were isolated from galleries and adults of T. minor and T. yunnanensis infesting P. yunnanensis (Table 2). Ophiostoma tingens was also reported from T. minor infesting P. yunnanensis in Yunnan (Pan et al. 2017).
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.
Sporothrix macroconidia was found associated with T. yunnanensis infesting P. yunnanensis and with T. brevipilosus infesting P. kesiya. The other four similar species have very different ecology and known geographic distributions. Sporothrix fumea was isolated from Eucalyptus cloeziana infested by Phoracantha beetles in South Africa (Nkuekam 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.
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 Tomicus yunnanensis and T. minor in Pinus yunnanensis.
The hosts and geographic distributions of S. pseudoabietina and S. abietina are also very different. Sporothrix pseudoabietina was found associated with T. 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, T. brevipilosus, T. minor and T. yunnanensis, inhabiting 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. macroconidia. A single strain remained unnamed.
The global ophiostomatoid fungal communities, associated with these three Tomicus species in pine forest, were dominated by far by three species, which are, in decreasing order of isolates, O. canum, O. brevipilosi and O. minus. Furthermore, these three ophiostomatoid species are not equally associated with the three Tomicus 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 galleries and adults of T. 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 the dominant species associated with T. 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 typographus, 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 T. 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 fitness of O. brevipilosi is therefore strongly linked to the presence of T. brevipilosus.
Ophiostoma brevipilosi was described originally from Yunnan, based on six strains, all isolated from T. 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 T. 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 T. 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 TUB2-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 translating 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 T. minor and of T. brevipilosus yielded less species; five species were isolated from T. minor, none of which was associated exclusively with this beetle and three species from T. brevipilosus, of which one was exclusive, O. brevipilosi. Five species are shared by both T. yunnanensis and T. minor and two species by both T. 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).
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' Paine 1999, Six 2012), while others can be associated with many vectors (Kostovcik et al. 2014). The species-specific associations include, for instance, Ips typographus and Endoconidiophora polonica, I. cembrae and End. laricicola (Harrington et al. 2002) or I. 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 T. 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 T. 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 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 ophiostomatoid species to both indigenous pines, except for L. yunnanense (Liao andYe 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 Tomicus species. 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 T. yunnanensis, T. 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 T. yunnanensis and the poorest in the galleries and adults of T. brevipilosus.
Three species, namely O. brevipilosi, O. canum and O. minus, dominate the ophiostomatoid communities; each is associated predominantly with one species of Tomicus, namely T. brevipilosus, T. 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 Tomicus spp. in other neighbouring 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.