Ascomycetes from the Qilian Mountains, China – Hypocreales

Abstract To investigate fungi from the Qilian Mountains in Gansu Province, ascomycetous specimens were collected and hypocrealean fungi were examined. Eighteen species belonging to six genera in the families Hypocreaceae and Nectriaceae were identified, including 11 species of Hypomyces and Trichoderma in Hypocreaceae and seven species of Nectria, Stylonectria, Thelonectria, and Thyronectria in Nectriaceae. Among them, Stylonectria qilianshanensis and Trichoderma gansuanum are new to science. DNA sequence analyses of combined ACL1, ITS, RPB2, and TEF1 regions confirmed their taxonomic placements. Morphological distinctions between the new species and their close relatives are discussed. Hypomyces tremellicola is reported for the first time in China.


Introduction
The Qilian Mountains is located across the northeastern Qinghai and western Gansu provinces (35°50'-39°19'N, 94°10'-103°04'E) at elevations ranging from 4000 to 6000 m. The area has a temperate climate with mean annual precipitation around 400 mm. Forests are composed mainly of mixed broad-leaf and coniferous trees. The Qilian Mountain National Nature Reserve, where ascomycetes were surveyed, is ex-tremely diverse in climate, vegetation, and geographic structure. Previous investigations of fungal resources have mainly focused on Basidiomycota (Gui et al. 2010;Xi et al. 2011). Our understanding of ascomycetes of the region needs to be broadened.
The order Hypocreales includes about 2700 species in 240 genera, which are divided into 12 families (Rossman et al. 1999;Lumbsch and Huhndorf 2007;Kirk et al. 2008;Lombard et al. 2015;Maharachchikumbura et al. 2016;Sun et al. 2017;Zhuang and Zeng 2017). Hypocrealean collections from the Qilian Mountains belong to the families Hypocreaceae and Nectriaceae. These families are ubiquitous in nature and exhibit very high species diversity in temperate and tropical regions (Rossman 1996;Rossman et al. 1999). They are economically important in fields of industry, environment protection, and agriculture. For instance, some species of Trichoderma Pers. play vital roles in production of industrial enzymes and antibiotics (Jangiret et al. 2017), while several species of Hypomyces (Fr.) Tul. & C. Tul. are pathogens of cultivated mushrooms (Tamm and Põldmaa 2013). Some members of Nectria (Fr.) Fr., Thelonectria P. Chaverri & C. Salgado, and Thyronectria Sacc. cause Abies and Rubus cankers (Hirooka et al. , 2012Salgado-Salazar et al. 2015). Therefore, discovery of fungi in Hypocreales is of theoretical and practical importance. Improvement and updating our knowledge of the group will provide useful information about sustainable utilization and conservation of natural resources.
Studies on fungi of this group in China dates back to 1895 when Trichoderma cornu-damae (Pat.) Z.X. Zhu & W.Y. Zhuang (as Hypocrea cornu-damae Pat.) was reported on rotten wood in Sichuan Province (Patouillard 1895). Early studies were initiated by Teng (1934Teng ( , 1935Teng ( , 1936Teng ( , 1963, and recent studies are summarized by Liang (2007), Zhuang (2013), and Zhuang and Zeng (2017). A survey of ascomycetes in the Qilian Mountains was carried out in 2018. A total of 67 specimens were examined in this study. Eighteen taxa belonging to seven genera were identified, including 11 species of Hypomyces and Trichoderma in Hypocreaceae, and seven of Nectria, Thelonectria, Thyronectria, and Stylonectria Höhn. in Nectriaceae. Stylonectria qilianshanensis and Trichoderma gansuanum are described and illustrated as new species. Hypomyces tremellicola is reported for the first time from China.

Sampling and morphological studies
Specimens were collected from the Qilian Mountains in Gansu Province, and they are deposited in the Herbarium Mycologicum Academia Sinica (HMAS). Cultures are kept in the State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences. The methods of Luo and Zhuang (2010) and Põldmaa et al. (2019) were followed for morphological observations. The ascomatal wall reactions to 3% potassium hydroxide (KOH) and 100% lactic acid (LA) were tested. To observe microscopic characteristics of perithecial walls, sections were made with a freezing microtome (YD-1508-III, Jinhua, China) at a thickness of 6-8 μm.
Cotton blue lactophenol solution and lactic acid solution were used as mounting media for examinations of anatomical structures and measurements of perithecia, asci, and ascospores. Photographs were taken with a Leica DFC450 digital camera (Wetzlar, Germany) attached to a Leica M125 stereomicroscope (Milton Keynes, UK) for gross morphology, and a Zeiss AxioCamMRc 5 digital camera (Jena, Germany) attached to a Zeiss Axio Imager A2 microscope (Göttingen, Germany) for anatomy. Measurements of individual structures were based on n = 30, except as otherwise noted. The culture of Hypomyces tremellicola was isolated from conidia found on the surface of the host. To determine colony features and growth rates, strains were grown on malt extract agar [MEA,2% (w/v)

Sequence alignment and phylogenetic analyses
Newly obtained sequences and those retrieved from GenBank are listed in Tables 1 and 2, respectively. The sequences were assembled, aligned and the primer sequences were trimmed using BioEdit 7.0.5 (Hall 1999), and converted to NEXUS files by ClustalX 1.83 (Thompson et al. 1997). The aligned sequences were combined in BioEdit and analyzed with Bayesian inference (BI) and maximum parsimony (MP) methods to determine the phylogenetic positions of the new species. The MP analysis was performed with PAUP 4.0b10 (Swofford 2002) using 1000 replicates of heuristic search with random addition of sequences and subsequent TBR (tree bisection and reconnection) branch swapping. Topological confidence of the resulting trees was tested by maximum parsimony bootstrap proportion (BP) with 1000 replications, each with 10 replicates of random addition of taxa. The Bayesian inference (BI) analysis was conducted by  (Nylander 2004). Four Markov chains were run simultaneously for 1000000 generations with the trees sampled every 100 generations. A 50% majority rule consensus tree was computed after excluding the first 2500 trees as 'burn-in'. Bayesian inference posterior probability (PP) was determined from the remaining trees. Branch support measures were calculated with 1000 bootstrap replicates. Trees were examined by TreeView 1.6.6 (Page 1996). The BIPP greater than 90% and MPBP greater than 70% were shown at the nodes.

Results
To determine taxonomic positions of the Hypomyces collections, sequences of ITS and 28S rDNA were searched against the NCBI GenBank database using BLASTN. Sequence comparisons showed that HMAS 247843 shares 99% sequence similarity with H. tremellicola, which confirmed its taxonomic position in the genus.
To place the Stylonectria specime n within a phylogenetic context, sequences of ACL1, ITS, and RPB2 regions from 15 species of the genus and relatives were analyzed using BI and MP methods. Clonostachys rosea (Preuss) Mussat and Trichoderma parareesei Atan., Jaklitsch, Komoń-Zel., C.P. Kubicek & Druzhin. were used as outgroup taxa. The partition homogeneity test (PHT) (P = 0.01) indicated that the individual partitions were not highly incongruent (Cunningham 1997), the three loci were thus combined for phylogenetic analyses. The combined datasets include 2197 characters, of which 964 were constant, 310 were variable and parsimony-uninformative and 923 were parsimony-informative. The MP analysis resulted in two most parsimonious trees (tree length = 4081, CI = 0.5285, HI = 0.4715, RI = 0.4459, RCI = 0.2357) with similar topology. The final matrix was deposited in TreeBASE with accession no. S25189. The BI tree is shown in Figure 1. The MP tree is similar to that of the BI tree in topology. HMAS 255803 was associated with other Stylonectria species forming a highly supported monophyletic group (BIPP/MPBP = 100%/100%), which confirmed its taxonomic position in the genus.
Notes. Hypomyces tremellicola is a new record for China. This species was originally described as Hypocrea tremellicola Ellis & Everh. (Ellis and Everhart 1892), and later transferred to Hypocreopsis P. Karst. (Seaver 1910) and Nectriopsis Maire (Gams and Zaayen 1982). Samuels (1976) redescribed the species and assigned it to Hypomyces. It usually grows on Crepidotus spp., and less frequently on Polyporus spp. and Pleurotus spp. The shape and size of conidia and chlamydospores of the Chinese material match well with the description provided by Zare and Gams (2019). Sequence comparisons showed that 4 bp and 1 bp divergences existed in ITS and 28S rDNA between the Chinese material (HMAS 247843) and a collection from Germany (CBS 441.65). We treat them as infraspecific variations. Distribution. China, United States.

Discussion
The genus Stylonectria, typified by S. applanata, was established as a monotypic genus by Höhnel (1915). It was included in Nectria by Booth (1959) and then treated as a synonym of Cosmospora Rabenh. by Rossman et al. (1999). Gräfenhan et al. (2011) (Zhang and Zhuang 2003). For some nectriaceous fungi, substrate type was considered of taxonomic importance (Zeng and Zhuang 2016). Species of Stylonectria are fungicolous and sometimes host-specific. For example, S. applanata is known only from Melogramma bulliardii Tul. & C. Tul. on Corylus avellana, S. carpini is restricted to a pyrenomycete on Carpinus, and S. wegeliniana colonizes solely on Hapalocystis bicaudata Fuckel on Ulmus glabra (Gräfenhan et al. 2011;Lechat et al. 2015). Similarly, S. qilianshanensis occurs on decayed fruit bodies of an ascomycete on the bark of Picea asperata. However, S. norvegica and S. purtonii have slightly wider host ranges. The former occurs on pyrenomycetes on Betulaceae and Fagaceae, while the latter is found on pyrenomycetes on coniferous trees. Stylonectria is currently a poorly known genus. Further investigations may provide useful information about its selection of hosts or substrates.
Gross morphology like stroma, ascospore features and asexual states were stressed for generic delimitations of the hypocrealean fungi. Species of Hypocreaceae possessing clavate to cylindrical, fleshy, bright-colored stromata were previously accommodated in Podostroma P. Karst. (Karsten 1892; Rossman et al. 1999). The accumulated molecular evidence argued that shape of fruit body is not phylogenetically distinctive within genus. Chamberlain et al. (2004) then synonymized Podostroma with Hypocrea Fr. (= Trichoderma). The diagnostic characteristics for Trichoderma include substrate, fruit body gross morphology, anatomy, extent of fertile region, surface pigmentation of stromata, and ascospore shape, size and ornamentation (Chamberlain et al. 2004). The genera Aphysiostroma Barrasa, A.T. Martinez & G. Moreno, Pseudohypocrea Yoshim. Doi, and Sarawakus Lloyd, which possess gliocladium-, trichoderma-and verticillimlike asexual states, were also synonymized with Trichoderma (Jaklitsch et al. 2014;Jaklitsch and Voglmayr 2015;Zeng and Zhuang 2017). The taxonomic position of T. gansuanum is confirmed by the combined sequence analyses of RPB2 and TEF1 regions and morphological characteristics, such as the stipitate, clavate, upright stromata, cylindrical asci, and disarticulating ascospores. A few stipitate species of Trichoderma have not been cultured or linked to asexual states (Chamberlain et al. 2004). Knowing the whole fungus is surely our future goal.