Ophiostomatoid fungi associated with mites phoretic on bark beetles in Qinghai, China

Bark beetle galleries are complex ecosystems where many microbes and other arthropods co-exist with the beetles. Fungi isolated from these galleries are often referred to as ‘beetle associates’, but the nature of these associations are poorly understood. The possibility that many of these fungi might in fact be mite associates is often overlooked. Several recent studies explored the diversity of fungi from conifer-infesting bark beetles and their galleries in China, but only one study considered phoretic mites and their fungi from conifer-infesting bark beetles in Yunnan, southwestern China. We studied the mites and fungi from galleries of four spruce-infesting bark beetle species in the high altitude forests of Qinghai province, western China. Mites were identified based on morphological characteristics, and fungi based on DNA sequences of four gene regions. In total, 173 mite individuals were collected belonging to 18 species in 11 genera. A total of 135 fungal isolates were obtained from the mites, representing 14 taxa from the Ophiostomatales. The most frequently isolated fungus was Ophiostoma nitidum, which represented 23.5% of the total isolates. More fungal species were found from fewer mites and bark beetle species than from the study in Yunnan. Although we could not elucidate the exact nature of interactions between mites and their fungi, our results re-enforce that these organisms should not be ignored in pest risk assessments of bark beetles, that often focus only on the beetles and their fungi. Three new species are described: Grosmannia zekuensis, O. manchongi, and O. kunlunense spp. nov., and our data revealed that O. typographi, recently described from China, is a synonym of O. ainoae.


INTRODUCTION
Mites (Arachnida, Acari) are commonly associated with bark beetles (Insecta, Coleoptera, Scolytinae) and their galleries (Hofstetter et al. 2015). Because of their small size, these mite species rely on the bark beetles for dispersal between plant hosts (Hofstetter et al. 2013). To date, about 270 mite species have been identified as associates of a limited number of bark beetle species, and many more are likely to be discovered (Hofstetter et al. 2013(Hofstetter et al. , 2015. The ecological roles of very few bark beetle-associated mites are well understood, but the different classes of feeding behavior (Hofstetter et al. 2013) suggests that their impacts in gallery ecosystems could be broad. Some of these mites, such as Pyemotes dryas, predate upon or parasitize the eggs and early larval instars of bark beetles (Wegensteiner et al. 2015) and thus have a direct effect on population dynamics of the beetles. However, the impacts of many mites on bark beetle behavior and population growth are indirect. A few species are known to be predators of nematodes, while many of the mite species are omnivores and feed on fungi, nematodes and dead arthropods in the galleries. Other species are exclusively mycetophagous, utilizing fungi as their only source of nutrition (Hofstetter et al. 2013).
It appears that some mycetophagous mites have preference for one or a few specific species of fungi. One such an example is Tarsonemus krantzi, associated with the southern pine beetle, Dendroctonus frontalis, that vector and feeds on Ophiostoma minus . Other mites such as Histiogaster spp. are generalists that can feed on several different fungal species (Hofstetter and Moser 2014). The most common fungi associated with bark beetles and their mites are the ophiostomatoid fungi (Hofstetter and Moser 2014). This is a polyphyletic group of fungi that includes several genera in Microascales and Ophiostomatales, that are characterized by spores produced in sticky droplets to facilitate dispersal by bark beetles and mites . Many bark beetles have specialized structures known as mycangia in which to carry the spores of these fungi (Six 2012). In the case of mites, most fungi appear to be vectored on their exterior surfaces (Moser et al. 1989b), but some mite species have evolved specialized structures known as, sporothecae, to ensure the successful transmission of specific fungal symbionts between hosts (Moser 1985).
Although the impact of most fungi on the fitness of the mites or their beetle vectors remains largely unknown, the three-way interaction between beetles, mites and fungi, has been elucidated in the case of the southern pine beetle, Dendroctonus frontalis, and its associated Tarsonemus mites. The larvae of D. frontalis are obligately dependent on two fungal species, Ceratocystiopsis ranaculosus and Entomocorticium sp. A, for nutrition . These fungi are outcompeted by Ophiostoma minus (Barras 1970;Bridges 1983;Goldhammer et al. 1990), the nutritional fungal mutualist of Tarsonemus mites, when the mites are present in the galleries of D. frontalis (Hofstetter et al. 2013). As a result of these interactions, large populations of Tarsonemus mites can suppress the reproduction of D. frontalis (Hofstetter et al. 2006).
The relationships between mites and their beetle vectors are generally unspecific, and a single species of mite can use many different beetle species as vector in order to reach its tree hosts (Hofstetter et al. 2013). Similarly, many ophiostomatoid fungi are promiscuous in their association with beetle species, and a single beetle species can vector a number of different fungal species (Kirisits 2004;Linnakoski et al. 2012;Taerum et al. 2013). In addition, one mite species can carry any of a number of ophiostomatoid fungi (Chang et al. 2017). It thus becomes extremely difficult to resolve specificity between any of these organisms. However, where galleries of different bark beetle species might be restricted to different parts of a specific tree and thus not overlap, it has been suggested that mites might facilitate the movement of fungal species between galleries of different beetle species (Chang et al. 2017).
There are several serious tree pathogens amongst the ophiostomatoid fungi. These include the well-known Dutch Elm Disease fungi, Ophiostoma ulmi and two varieties of O. novo-ulmi (Brasier 1990(Brasier , 1991. These pathogens are vectored by Scolytus bark beetles (Webber 2004), but it has been shown that phoretic mites also vector the pathogens and might contribute to the spore load and the high efficiency of S. scolytus in spreading Dutch elm disease (Moser et al. 2010). Leptographium wingfieldii is a mildly pathogenic ophiostomatoid fungus vectored by the pine shoot beetle, Tomicus piniperda, in its native range in Europe (Solheim 1991). The beetle was first detected in the USA in the early 1990's, and it was later shown that L. wingfieldii was introduced with the beetle, and that the fungus has subsequently became an associate of two native north American beetles, Dendroctonus valens and Ips pini (Jacobs et al. 2004). Although the mechanism by which L. wingfieldii was transferred between the beetle species was not considered (Jacobs et al. 2004), it is likely that mites facilitated the transfer. Such novel associations between ophiostomatoid fungi, bark-or ambrosia beetles and trees, can pose serious threats to both natural and commercial forests, as well as tree crops. The role of mites in the establishment of these associations are poorly studied, most probably underestimated, and can only be understood if their associations with fungi are explored in natural ecosystems.
The taxonomy of Ophiostomatales was revised by  who considered all published ribosomal large subunit (LSU) and internal transcribed spacer (ITS) sequences. They recognized six genera and 18 species complexes in the order, including Ophiostoma, Raffaelea, Ceratocystiopsis, Fragosphaeria, Graphilbum, and Leptographium sensu lato. In a subsequent paper De Beer et al. (2016a) elevated the S. schenckii -O. stenoceras complex to genus level, and re-instated the name Sporothrix for this group. Three smaller, novel genera had also been described recently in the order, Hawksworthiomyces (De Beer et al. 2016b), Aureovirgo (Van Der Linde et al. 2016) and Afroraffaelea (Bateman et al. 2017). For the purpose of the present study, we define Ophiostoma sensu stricto in agreement with De Beer et al. (2016a), that then includes the O. ulmi-, O. piceae-, O. ips-, and O. clavatum species complexes. A number of Ophiostoma species group outside Ophiostoma s. str., and their position remain unresolved. For the present they are included next to Ophiostoma s. str. in a more loosely defined Ophiostoma sensu lato. Leptographium s. lat. incorporates Leptographium s. str. and the Grosmannia penicillata complex as defined by  and Yin et al. (2020).   To date, the majority of studies on interactions between fungi, bark beetles and mites have been conducted on Dendroctonus, Ips and Dryocoetes spp. in North America (Klepzig and Hofstetter 2011;Hofstetter et al. 2013Hofstetter et al. , 2015Hofstetter and Moser 2014), and Scolytus, Ips and Pityokteines spp. in Europe (Levieux et al. 1989;Moser et al. 1989aMoser et al. , 2005Moser et al. , 2010Linnakoski et al. 2016b). In southern Africa, ongoing studies focus on the interactions between mites and ophiostomatoid fungi in infructescences of Protea spp. (Roets et al. 2007(Roets et al. , 2009(Roets et al. , 2011. There have been only two studies reporting on the fungal associates of mites form east Asia. In these cases, Moser et al. (1997) reported on the fungal associates of mites on Ips typographus in Japan, and Chang et al. (2017) described fungi from mites associated with various conifer-infesting bark beetles in Yunnan, China.
In recent years, 98 ophiostomatoid species, including 49 new species, have been reported in association with bark beetles from China (Table S1) (Lu et al. 2009a(Lu et al. , 2009bPaciura et al. 2010aPaciura et al. , 2010bZhou et al. 2011Zhou et al. , 2013Taerum et al. 2013;Yin et al. 2015Yin et al. , 2016Yin et al. , 2019Yin et al. , 2020Wang et al. 2016Wang et al. , 2018Wang et al. , 2019Wang et al. , 2020Chang et al. 2017Chang et al. , 2019Liu et al. 2017). The study by Chang et al. (2017) was the first to report ophiostomatoid fungi associated with phoretic mites. They reported 11 species from mites associated with bark beetles infesting Pinus kesiya, of which four were described as new species. Three of the studies from China, included isolates from Qinghai province (Yin et al. 2016(Yin et al. , 2019(Yin et al. , 2020. This province, which is located on the Qinghai-Tibetan Plateau, is one of the world's biodiversity hotspots because of its diverse landscapes, and complex geological and climatic history (Li et al. 2012). Yin et al. (2016) decribed five new Ophiostoma spp. from four spruce-infesting bark beetles, two of which are Ips spp. that cause severe damage to spruce trees are in this area (Liu et al. 2008). In addition, two Leptographium (Yin et al. 2019) and four Grosmannia spp. (Yin et al. 2020) were described from Polygraphus poligraphus and Ips shangrila, also attacking spruce. Apart from these 11 species, no other ophiostomatoid fungi have been reported from Qinghai. Furthermore, nothing is known regarding the fungal associates of phoretic mites on spruce-infesting beetles in China. In this study, we addressed the following questions: 1) which mite species are associated with spruceinfesting bark beetles in Qinghai, and 2) which species of ophiostomatoid fungi are associated with these mites?

Collection of mites and fungi
A survey was conducted on Picea crassifolia and Picea purpurea in July 2010, during the flight period of bark beetles, in the Maixiu and Xianmin forest farms in Qinghai province, China. Bark beetle galleries were collected and stored in re-sealable plastic bags at 4°C until isolations could be made. Living mites were collected from the galleries under a dissecting microscope. Each individual mite was placed on a separate Petri dish containing malt extract agar (MEA, 20 g Difico agar, 20 g Difico BactoTM malt extract [Becton, Dickinson & Company], 1 L deionized water) medium. After the plates were sealed, the mites were allowed to crawl over the plates for 24 h. The mites were then removed and stored in 1.5 mL Eppendorf tubes containing 75% Ethanol for later identification by Dr. E.A. Ueckermann (Plant Protection Research Institute, Agricultural Research Council, South Africa).
The MEA plates were incubated at 20°C until fungal growth was evident. The hyphal tips of colonies were transferred to fresh MEA plates to obtain pure cultures. All isolates used in this study were deposited into the Culture Collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, Republic of South Africa. Isolates representing types of new species were deposited in the culture collection (CBS) of the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands.

DNA sequencing and phylogenetic analyses
Isolates were grown on 2% MEA medium. DNA was extracted using PrepMan ultra sample preparation reagent (Applied Biosystems, Foster City, CA) following the manufacturer's recommendations. The internal transcribed spacer regions 1 and 2 (ITS), including the 5.8S region, were amplified using the primers ITS1F and ITS4 (White et al. 1990;Gardes and Bruns 1993), the β-tubulin (BT) gene was amplified using the primer pair of Bt2a and Bt2b (Glass and Donaldson 1995), and the elongation factor 1α (EF) gene was amplified using the primer pair of EF2F (Marincowitz et al. 2015) and EF2R (Jacobs et al. 2004). In addition, the nuclear large subunit (LSU) was amplified with the primer pair LR0R and LR5 (Vilgalys and Hester 1990) for fungi that reside in Leptographium sensu lato. PCR and sequencing were conducted following the protocols described by Duong et al. (2012).
The sequences obtained with the forward and reverse primers were aligned and contigs constructed using the program Geneious pro v. 7.1.4 (Biomatters, Auckland, New Zealand). All sequences obtained in this study were deposited in GenBank. BLAST searches of the ITS sequences were conducted in NCBI GenBank for preliminary identifications. Based on the BLAST results, sequence data for other markers were separated according to the relevant species complexes. For taxa residing in Leptographium s. lat., the ITS2-LSU regions were used to determine generic placement, unlike the case for Ophiostoma spp. where the ITS1-ITS2 regions were used for this purpose. The BT and EF data sets were analyzed separately for each species complex. Alignments were made using an online version of MAFFT v. 7 with default settings (Katoh and Standley 2013). All aligned sequence datasets were submitted to TreeBase (No. 24829).
Phylogenetic analyses including maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) were conducted for all datasets. The best substitution models for each data set were determined using jModelTest v. 2.1.6 (Darriba et al. 2012) on the CIPRES Science Gateway v. 3.3 (Miller et al. 2010). ML analyses were conducted using RaxML v. 8.2.4 on the CIPRES Science Gateway v. 3.3 (Stamatakis 2014) with default GTR substitution matrix and 1000 rapid bootstraps. MP analyses were performed using PAUP v. 4.0b10 (Swofford 2002), gaps were treated as a fifth character. BI analyses were conducted using MrBayes v. 3.2.6 (Ronquist et al. 2012) on the CIPRES Science Gateway v. 3.3. Four MCMC chains were run from a random starting tree for 5 million generations and trees were sampled every 100th generation. 25% of trees sampled were discarded as burn-in and the remaining trees were used to construct majority rule consensus trees.

Growth studies
Mycelium-covered agar plugs were transferred from the actively growing margins of one-week-old cultures and placed at the centers of 90 mm Petri dishes containing 2% MEA. The optimal temperatures for growth were determined at temperatures ranging from 5 to 35°C at 5°C intervals and there were three replicate plates for each temperature. Cultures were incubated in the dark. Colony diameters were measured every 2 days until hyphae reached the edges of the Petri dishes, at which point the temperatures for optimum growth were noted.

Morphological studies
Asexual and/or sexual structures were mounted in lactophenol on glass slides, covered with coverslips and examined with a Zeiss Axioskop 2 Plus compound microscope or a Zeiss Discovery V12 dissection microscope with an Axiocam digital camera (Axiovision 3.1) (München-Hallbergmoos, Germany). Fifty measurements were made for each taxonomically informative structure. The measurements were given in the format (minimum-) mean minus standard deviation-mean plus standard deviation (−maximum).

Frequency of isolation
The following formula was used to calculate the frequencies of isolation of the ophiostomatoid species: F = (NF/NT) × 100, where F represented the frequency of isolation (%), NT represented the total number of isolates obtained, and NF represented the number of isolates for each particular taxon.

Collection of mites and fungi
In total, 173 mite individuals representing 18 mite species residing in 11 genera were collected from galleries of four bark beetle species on two host tree species (Table 1). The bark beetles were Dendroctonus micans, Ips shangrila, Ips nitidus and Polygraphus poligraphus. All D. micans, I. nitidus and P. poligraphus were collected from Pi. crassifolia, and I. shangrila was collected from Pi. purpurea. Three mite species were associated with D. micans, 13 mite species with I. nitidus, five mite species with I. shangrila, and three mite species with P. poligraphus. Most mite species were found only in the galleries of a single bark beetle species. Exceptions were for Insectolaelaps sp. 1, Uropodoidea sp. 4, Uropodoidea sp. 6, and Winterschmidtiidae sp., which were found in the galleries of more than one beetle species (Table S2).
In total, 135 ophiostomatoid fungal isolates were obtained from 65 mite individuals (Table S3). Seventy isolates were collected from 13 mite species in galleries of I. nitidus, 33 isolates were collected from three mite species in galleries of D. micans, 19 isolates were collected from five mite species in galleries of I. shangrila, and 13 isolates were collected from three mite species in P. poligraphus. Twenty-eight isolates were collected from one mite species in the family Winterschmidtiidae and 23 isolates collected from Uropodoidea sp. 4. More than 10 isolates were collected from each of Insectolaelaps sp. 2, Insectolaelaps sp. 1 and Uropodoidea sp. 6.

DNA sequencing and phylogenetic analysis
Based on analysis of ITS and ITS-LSU sequence data, of the total 135 isolates collected in this study, 87 isolates resided in Ophiostoma sensu stricto (Fig. 1), and the remaining 48 isolates resided in Leptographium s. lat. (Fig. 2). Most of the isolates belonging to Ophiostoma s. str. resided in three species complexes namely the O. piceae-, O. clavatum-and O. ips species complexes, while most of the isolates belonging to Leptographium s. lat. resided in the Grosmannia penicillata species complex. Based on the availability of sequence data from previously studies, datasets of different protein coding gene regions were compiled and analysed separately for different species complexes. Phylogenetic analyses of these datasets separated the isolates into 14 distinct taxa ( Table 2), 11 of which belonged to previously described species and three represented novel species.
In Ophiostoma s. str., two taxa (Taxa 1 and 4) represented by four and three isolates respectively, grouped peripheral to the O. piceae complex in the ITS tree (Fig. 1). Analyses of BT and EF sequences data (Fig. 3) showed that Taxon 1 belong to O. tetropii, and isolates of Taxon 4 formed a distinct, well supported clade (with 100% ML bootstrap support and BI posterior probabilities larger than 0.9 for both   BT and EF gene regions) which was separated from all previously described species, and thus this taxon represented a novel species. Taxa 2 and 3, represented by 32 and 11 isolates respectively, grouped with O. nitidus and O. qinghaiense in O. piceae complex. Taxa 5 and 6 grouped in the O. clavatum complex (Fig. 1) and were represented by ten and 11 isolates respectively. Based on the BT and EF sequence data (Fig. S1) the Taxon 5 isolates grouped in a single clade with the ex-type isolates of both O. ainoae and O. poligraphi, suggesting that these represented a single species. Taxon 6 isolates grouped with sequences of O. shangrilae. Taxon 7 resided close to and Taxon 8 within the O. ips complex (Fig. 1) and were represented by ten and six isolates respectively. Based on ITS and BT data (Fig. 4) isolates of Taxon 7 formed a distinct and well supported clade (with 97% ML bootstrap support for ITS, 100% ML bootstrap support for BT, and BI posterior probabilities larger than 0.9 for both ITS and BT gene regions) which was closest to, but clearly distinct from O. japonicum, and thus this taxon represented a novel species. Taxon 8 isolates grouped in a monophyletic lineage with several isolates of O. bicolor.
In Leptographium s. lat., Taxon 9 represented by only one isolate grouped in Group A of Leptographium s. lat. together with L. pineti and L. ningerensis (Fig. 2) and BT and EF sequence analyses confirmed this Taxon was conspecific with L. ningerensis (Fig. S2). Taxon 10 grouped in the L. olivaceum complex (Fig. 2) and BT and EF sequence analyses confirmed that this isolate represented L. breviuscapum (Fig. S2). Taxon 11 grouped peripheral to the G. penicillata complex with L. taigense (Fig. 2) and BT and EF sequences analyses confirmed the identity of the species as L. taigense (Fig. 5). Taxa 12, 13, and 14 grouped in the G. penicillata complex (Fig. 2) and based on BT and EF sequences (Fig.  5), Taxon 12 was identified as G. purpurea, and Taxon 14 was identified as G. xianmiense, while isolates of Taxon 13 formed a distinct and well supported clade (with ML bootstrap supports larger than 95%, and BI posterior probabilities larger than 0.9 for both BT anf EF gene regions), which was close to but different from G. purpurea, thus this taxon represented a novel species.

Frequencies of isolation
The most frequently collected mite species were Uropodoidea sp. 4 and Uropodoidea sp. 6 each of which represented 21.2% of all mites (Table 1). The next most frequently collected species was Insectolaelaps sp. 2 which represented 13.9% of the mites. A few species were collected at very low frequencies, such as Uropodoidea sp. 7 and Uropodoidea sp. 8 that only represented 0.6% of the collections. The most frequently isolated ophiostomatoid fungi were O. nitidum (Taxon 2) and L. taigense (Taxon 11), which represented 23.5 and 22.8% of the total isolates respectively (Table S3). This was followed by O. qinghaiense (Taxon 3, 8.1%) and O. shangrilae (Taxon 6, 8.1%), and O.
ainoae (Taxon 5, 7.4%) and Taxon 8 (7.4%). The fungi with the lowest frequency of isolations were L. breviuscapum (Taxon 10) and L. ningerensis (Taxon 9), both of which represented 0.7% of total isolates. The remaining fungi were found at frequencies lower than 5%.  The number of fungal isolates collected from different mite species differed substantially (Table S3). About 20% of the fungi were isolated from mites in the family Winterschmidtiidae. Twenty-four isolates collected from Uropodoidea sp. 4 represented 17% of the total isolates. This was followed by 12.1% of the isolates collected from Insectolaelaps sp. 2, 10% isolates collected from Insectolaelaps sp. 1, 10.6% isolates collected from Uropodoidea sp. 6 ( Table S3).
The 33 isolates collected from the mites in D. micans galleries represented 23.4% of the total number of isolates, while 75 isolates from the mites in I. nitidus galleries represented 53.2%, 19 isolates from the mites in I.
shangrila galleries represented 14.2%, and the remaining 13 isolates from mites in P. poligraphus galleries represented 9.2% of the isolates.

TAXONOMY
Based on the phylogenetic analyses of different gene regions, as discussed above, three out of the total 14 taxa identified in this study represented novel species, and descriptions for these novel species are provided below. Phylogenetic analyses of BT and EF gene regions (Fig. S1) also indicated that O. ainoae and O. poligraphi belonged to the same species, thus Ophiostoma poligraphi is synonymized with Ophiostoma ainoae. MycoBank MB 827335 (Fig. 6) Etymology. Name refers to the Kunlun mountains, which is one of the longest mountain ranges in Asia, that ends in Qinghai province from where this fungus was first isolated. Description: Sexual morph not observed. Asexual morph pesotum-like, occurring singly or in groups of up to 15, macronematous, synnematous, erect, (511-) 705-1729.5 (− 1301) μm long, including condiogenous apparatus. Conidia hyaline, 1-celled, smooth, oblong, clavate or obovoid (3.5-) 3-6 (− 8) × 1.5-2(− 2.5) μm, accumulating in a white, gelatinous mass at the apex of the synnema.

Diagnosis
Culture characteristics: Colonies hyaline or dark brown when synnemata form. Mycelium superficial on the agar. Pesotum-like asexual morph dominant in the cultures. Optimal temperature for growth 20°C, reaching 56.6 mm diam in 10 d. No growth observed at 5°C or 30°C and above.
Notes: When BT and EF sequences produced for several isolates obtained in the present study were analysed together with sequences of three O. ainoae isolates from the study of Linnakoski et al. (2016a) and two O. poligraphi isolates from the study of Yin et al. (2016), it became clear that the latter two groups of isolates represented geographically isolated populations of the same species. Sequences of our isolates did not group consistently with isolates in either of the two clades (Fig.  S1). For the BT region, our isolates grouped between the two 'species', while in the EF analyses, they all grouped with O. poligraphi. There are confirmed reports of O. ainoae from Ips typographus and Pityogenes chalcographus on Picea abies in Europe (Linnakoski et al. 2016a), while O. poligraphi was described from Polygraphus poligraphus and Dendroctonus micans on Pi. crassifolia in Qinghai (Yin et al. 2016). Our isolates were from mites on P. poligraphus and Ips nitidus, also from Pi. crassifolia in Qinghai, and this is consistent with the host and beetle vectors of both species. Although a sexual state has not been described for O. poligraphi, the broad synnemata of this species also correspond with those described for O. ainoae (Solheim 1986), supporting the synonymy of the two species.

DISCUSSION
We collected 173 mites representing 18 species from spruce-infesting bark beetles in Qinghai province, China. Uropodoidea sp. 4 and Uropodoidea sp. 6, were most abundant among the mites. We obtained 135 fungal isolates from 65 mite individuals. Based on DNA sequence data, 14 fungal species were identified, eight species of Ophiostoma and six of Leptographium s. lat. Among these species, three were recognized as novel taxa and were thus described and provided with names. Of the remaining 11 species, ten had previously been reported from China. Ophiostoma tetropii is reported for the first time from China. The only previous study to report on ophiostomatoid fungi associated with phoretic mites in China was conducted on mite associates of six pine-infesting bark beetles in Yunnan (Chang et al. 2017). The species diversity of mites collected in the present study from Qinghai was higher than that found in Yunnan (18 species vs. 13), despite the fact that only four beetle species were sampled in Qinghai as opposed to six in Yunnan. However, more mite individuals (173) were collected in Qinghai from two host trees than the 106 mites from only Pinus kesiya in Yunnan (Chang et al. 2017). Only Insectolaelaps sp. 1 was collected from both Qinghai and Yunnan. This species was the most frequently collected species in Yunnan and the fourth most frequently collected species in Qinghai, suggesting that it is widely distributed on conifer hosts in western China. The fact that only one species was shared between the two regions is perhaps not surprising because the mites were collected from different bark beetle species, different tree species, and different climatic zones. Together with the previous report from Yunnan (Chang et al. 2017), a total of 31 species of mites associated with ten bark beetles have now been reported from western China.  This number is much less than 270 mite species that have been reported in association with more than 110 bark beetle species from the many studies in North America and Europe (Hofstetter et al. 2015), 12 mite species were reported from Ips typographus in Japan, of which only three species did not occur in Europe (Moser et al. 1997). Because most of the mites in our study have not been identified to species level, and because our sampling strategy was aimed at isolating fungi and not only the collection of mites, it is not possible to compare the presence or absence of mite species with other studies. However, at a higher taxonomic level, the most frequent mite order associated with bark beetles was the Mesostigmata in both China and collections collectively from Europe and North America, where it is represented by 20 and 140 species respectively (Hofstetter et al. 2015). The fungal species diversity from mites in Qinghai was somewhat higher when compared with the diversity of fungal species from mites in Yunnan (Chang et al. 2017), with 12 species reported from Yunnan versus 14 species in Qinghai, although the total number of fungal isolates was smaller in Qinghai. The fungi collected in Qinghai all belonged to only two genera in the Ophiostomatales, Ophiostoma and Leptographium s. lat., while isolates from Yunnan belonged to five genera, four (Graphilbum, Leptographium s. lat., Ophiostoma and Sporothrix) in the Ophiostomatales, and Graphium in the Microascales. The fungal community collected from bark beetle-associate mites in Qinghai was very different to that in Yunnan. Only one fungal species, L. ningerensis, was shared between Qinghai and Yunnan.
A comparison of the fungal species from spruce infesting bark beetles in Qinghai (Yin et al. 2016) with those from mites in our study, revealed that eight species were found in both studies. These included: L. breviuscapum, G. purpurea, G. xianmiense, O. nitidum, O. qinghaiense, O. ainoae, O. shangrilae and O. tetropii. Not surprisingly, a large number of fungal species were also shared between galleries and mites in Yunnan province (Chang et al. 2017).
Apart from the three new species discovered in this study, we also collected and identified O. tetropii for the first time from China. This fungus was first described from spruce tree attacked by Tetropium sp. in Sweden (Mathiesen 1951), and was subsequently also reported from Picea abies attacked by Ips typographus and Pityogenes chalcographus in Finland (Linnakoski et al. 2010) and Picea rubens attacked by Tetropium fuscum in Canada (Harrison and Smith 2013). Its presence on mites in China is surprising because it was considered as an associate and indicator of beetles such as T. fuscum (Harrison and Smith 2013).

CONCLUSIONS
The results of this study conducted in China reaffirmed the findings elsewhere in the world showing that there are close associations between bark beetle-associated mites and ophiostomatoid fungi. The sampling area, beetle species and hosts considered was relatively limited. Yet many undescribed species of mites and various novel taxa in Ophiostoma and Leptographium emerged from the study. This reflects how little is