Rubroshiraia gen. nov., a second hypocrellin-producing genus in Shiraiaceae (Pleosporales)

Abstract Shiraiaceae is an important family in Pleosporales (Dothideomycetes), which includes medical fungi and plant pathogens. Two hypocrellin-producing taxa, Shiraia bambusicola and a novel genus Rubroshiraiagen. nov., typified by Rubroshiraia bambusae are treated in this article. Maximum likelihood analysis, generated via RAxML (GTR+G model), using a combined SSU, LSU, TEF1 and RPB2 sequence dataset, shows that Rubroshiraia is close to Shiraia and belongs to the family Shiraiaceae. Descriptions, illustrations and a taxonomic key are provided for the genera in Shiraiaceae. Rubroshiraia morphologically differs from Shiraia in having small and dark ascostromata and filiform ascospores. Production of the ascostromatal metabolites, hypocrellin A and B, were examined by HPLC and spectrophotometer. The content of hypocrellin A and B of specimen HKAS 102255 (R. bambusae) is twice that produced by HKAS 102253 (S. bambusicola). To clarify the relationship between R. bambusae and Hypocrella bambusae, type material of the latter was examined and provided the illustration.

Shiraia has previously been placed in several families, depending on the opinions of authors. Hennings (1900) considered Shiraia to have unitunicate asci and treated as a member in the family Nectriaceae Tul. & C. Tul. (Hypocreales, Sordariomycetes) when he established the genus. Based on its large and fleshy fruiting bodies, Shiraia was transferred to Hypocreaceae De Not by Saccardo (1902). Amano (1980) re-examined the type specimen and regarded Shiraia as having bitunicate asci and, hence, placed the genus in Pleosporaceae Nitschke (Pleosporales, Dothideomycetes). However, it was subsequently transferred to Dothideales, genera incertae sedis by Kirk et al. (2001).
Earlier classifications of Shiraia were based on morphological characters. The first attempt of DNA-based taxonomy (Cheng et al. 2004) confirmed that Shiraia belongs in Pleosporales and was phylogenetically close to species of Phaeosphaeriaceae M.E. Barr. Thus, Cheng et al. (2004) considered Shiraia as a member in Phaeosphaeriaceae. Liu et al. (2013) carried out significant studies on Shiraia taxonomy by re-examining the holotype and carrying out phylogenetic analysis, based on LSU sequence data. Liu et al. (2013) also designated an epitype of both sexual and asexual morphs and introduced Shiraiaceae in the Pleosporales.
Shiraia bambusicola has been reported as a pathogen on various bamboo species (Table 2) or as endophyte of bamboo culms (Morakotkarn et al. 2007(Morakotkarn et al. , 2008. The bamboo genus Brachystachyum Keng is significantly affected by S. bambusicola (Table 2; Lai and Fu 2000). The holotype of S. bambusicola was recorded from a Bambusa sp. (Liu et al. 2013). Shiraia bambusicola has also been recorded on several common bamboo genera, including Fargesia Franch., Phyllostachys Sieb. et Zucc., Pleioblastus Nakai and Indosasa Mcclure (Lai and Fu 2000;Li et al. 2009). However, these hosts need to be further verified.
A Chinese medical fungus named "Zhuhongjun" in Chinese, was identified as Hypocrella bambusae (Berk. & Broome) Sacc. by Liu (1978), based on its conspicuous and fleshy fruiting body. However, according to our knowledge, Zhuhongjun is similar to S. bambusicola and unrelated to Hypocrella. Therefore, the taxonomic status of this taxon needs to be clarified.
The monotypic genus Grandigallia, collected on Polylepis sericea Wedd. (Rosaceae), was introduced by Barr et al. (1987) with G. dictyospora M.E. Barr et al. as the type species. Grandigallia dictyospora was reported from Venezuela in a locality above 3,400 m and the fungus was found to produce large ascostromata (3-14 cm in diam.), with bitunicate asci and dictyospores (Barr et al. 1987).
In this study, ten specimens of S. bambusicola and a hypocrellin producing taxon ("Zhuhongjun" in Chinese) were collected from Yunnan Province in China. Morphological and phylogenetic studies were carried out to determine the taxonomic status of these taxa. Sequences from endophytic strains, named as Shiraia spp., were also downloaded from GenBank and included in the phylogenetic analyses. The metabolite content of hypocrellin extracted from the specimens was determined by HPLC (Chem 2012). Based on the morphology and phylogenetic analyses, the hypocrellin producing taxon "Zhuhongjun" is treated as a new genus in Shiraiaceae.

Collecting and examination of specimens
Bamboo culms with large, reddish to pale yellow ascostromata were collected from Yunnan, China and brought to the laboratory in 2017. Samples were examined following the methods described in Dai et al. (2017). Micro-morphological characters were examined and photographed by differential interference contrast (DIC), using a Leica DM2500 compound microscope with a Leica DMC4500 camera. Fruiting bodies were observed by stereomicroscopy using a Leica S8AP0

DNA extraction, PCR amplification and sequencing
The surface of fungal fruiting bodies was sterilised by 75% alcohol and rinsed three times in sterile water. The internal tissue with locules was cut into pieces and ground in a mortar into powder with liquid nitrogen. The powder was used to directly extract DNA with an OMEGA E.Z.N.A. Forensic DNA Kit, following the manufacturer's instructions. ITS5 and ITS4, NS1 and NS4 (White et al. 1990) and LROR and LR5 (Vilgalys and Hester 1990) primers were used for the amplification of internal transcribed spacers (ITS), small subunit rDNA (SSU) and large subunit rDNA (LSU), respectively. Translation elongation factor 1-α gene region (TEF 1-alpha) and RNA polymerase II second largest subunit (RPB2) genes were amplified by using EF1-983F and EF1-2218R (Rehner 2001), fRPB2-5f and fRPB2-7cr primers (Liu et al. 1999), respectively.
The final volume of the polymerase chain reaction (PCR) was prepared following Dai et al. (2017). The PCR thermal cycle programme of ITS, SSU, LSU, RPB2 and TEF 1-alpha genes amplifications were run under the same conditions as described in Dai et al. (2017). The quality of PCR products was checked by 1% Biowest agarose gel electrophoresis. Amplified PCR fragments were sequenced at Shanghai Majorbio Bio-Pharm Technology Co., Ltd. and BGI Tech Solutions Co., Ltd. (BGI-Tech), P.R. China. Generated new sequences of ITS, LSU, SSU, Rpb2 and TEF1 regions are deposited in GenBank (Table 4).  The holotype specimen is highlighted in bold. Abbreviations: HKAS: herbarium of Kunming Institute of Botany, Chinese Academy of Sciences.

Phylogenetic analysis
The BLAST searches in GenBank, using LSU and ITS sequence data were carried out to obtain the close strains. Additional sequences were downloaded from GenBank based on recent publications . Single gene sequence alignments were carried out with MAFFT v. 7.215 (Katoh and Standley 2013, http://mafft.cbrc.jp/alignment/server/index.html) and edited manually when necessary in BioEdit v. 7.0 (Hall 2004). The alignments of LSU, SSU, Rpb2 and TEF1 regions were combined in MEGA6 version 6.0 (Tamura et al. 2013).
Maximum-parsimony (MP) analyses were carried out in PAUP v. 4.0b10 (Swofford 2002) with 1000 replications. Maxtrees were set to 1000, branches of zero length were collapsed and all multiple equally most parsimonious trees were saved. The robustness of the most parsimonious trees was evaluated from 1000 bootstrap replications (Phillips et al. 2013).
Bayesian analyses were performed using MrBayes v. 3.0b4 (Ronquist and Huelsenbeck 2003). The model of evolution was performed using MrModeltest v. 2.2 (Nylander 2004). Posterior Probabilities (PP) (Rannala and Yang 1996;Zhaxybayeva and Gogarten 2002) were determined by Markov Chain Monte Carlo sampling (MCMC) in MrBayes v. 3.0b4 (Huelsenbeck and Ronquist 2001). Six simultaneous Markov chains were run for 1,000,000 generations and trees were sampled every 100 th generation. The burn-in was set to 0.25 and the run was automatically stopped when the average standard deviation of split frequencies reached below 0.01 .

HPLC profiling
Standards of hypocrellin A and hypocrellin B were purchased from Shanghai Tauto Biotech CO., Ltd. (http://www.tautobiotech.com) and used as received. Their purity is ≥ 98% (HPLC) and their structures are redrawn based on references (Wan and Chen 1981;Morakotkarn et al. 2008) and shown in Figure 1. The dry powder of ascostromata of S. bambusicola (HKAS102266) and "Zhuhongjun" (HKAS102270) was extracted followed the methods described by Stadler et al. (2001) and accurately weighed to 0.5 g and added to 25 ml of methanol and sonicated for 30 min. Semi-preparative HPLC was performed on an Agilent 1260 apparatus equipped with a UV detector and a CAPCELL PAK C18 (Agilent, 4.6 mm × 25 cm, 5 µm) column, with 38% solvent A: H 2 O + 0.5% formic acid; 62% solvent B: acetonitrile, isocratic elution, UV/Vis the detection in the range of 265 nm (Table 3). The UV-Vis spectra were recorded at room temperature on a Perkin-Elmer Lambda 900 spectrophotometer (Fig. 5).

Phylogeny
To clarify the family placement of newly established taxa, maximum likelihood phylogenetic analysis was generated from RAxML (GTR+G model), based on combined SSU, LSU, TEF1 and RPB2 sequences data (Fig. 2). The combined alignment comprised 4025 characters including gaps for 127 ingroup taxa and one outgroup taxon Dothidea insculpta (CBS 189.58). Based on the phylogenetic tree in Fig. 2, the new collections cluster within family Shiraiaceae with high bootstrap support (96/1.00 MLBS/BSPP) and emerge as two groups, which are S. bambusicola lineage and a new clade named as R. bambusae in this paper. Shiraia and Rubroshiraia have more or less similar ascostromata and both of them can produce the metabolite hypocrellins. However, they can be phylogenetically distinguished with high bootstrap support (100/1.00 MLBS/BSPP) (Fig. 2). Grandigallia has not been included in phylogenetic analysis as it is lacking sequences in the GenBank. However, the new taxa can be morphologically distinguished from it. Shiraiaceae is phylogenetically close with family Phaeosphaeriaceae in Pleosporales and this has been confirmed by Liu et al. (2013).
To clarify the relationship between endophytic strains named as shiraia-like (Shiraia spp.) and Shiraiaceae, a phylogenetic tree was constructed (RAxML (GTR+G model), based on combined LSU and ITS sequences data and compared. The combined alignment comprises 1442 characters including gaps for 57 ingroup taxa and one outgroup taxon Pleospora herbarum (CBS 191.86). Of the 1442 characters of the combined matrix, 1116 were constant and 220 were parsimony informative. The endophytic strains separated into two lineages (Group A and group B) forming at the base clade of Shiraiaceae (Fig. 3). Several strains in group A ca. JP7, JP93, JP232, JP256, SUPER-H168, A8 and ML-2004, isolated from bamboo tissue can produce hypocrellins in media (Lu et al. 2004;Morakotkarn et al. 2008;Liang et al. 2009;Cai et al. 2011;Zhang et al. 2014). However, no hypocrellins were detected from Group B, which included three Japanese strains viz. JP119, JP151 and JP185 (Morakotkarn et al. 2008).   (CBS 191.86). Ex-type or ex-epitype strains are in bold. Newly generated strains are in red.

Metabolites production
Stromatal extracts from specimens of S. bambusicola (HKAS102266) and R. bambusae (HKAS102270) contained high quantities of hypocrellin A (304.03 ng/ul and 790.86 ng/ul, respectively). Stromatal extracts from specimens of S. bambusicola contained 42.55 ng/ul hypocrellin B, whereas R. bambusae produces a higher quantity (204.60 ng/ul). The HPLC profiles of S. bambusicola and R. bambusae are depicted in Figure  4. The UV spectrum of the standards and of hypocrellin A and B from the samples (S. bambusicola HKAS 102253 and R. bambusae HKAS 102255) were recorded in alcohol and shown in Figure 5.  Ariyawansa et al. (2013). In previous studies, Shiraiaceae was closely related with Phaeosphaeriaceae and their distinction was questionable (Cheng et al. 2004, Liu et al. 2013). However, our multi-gene analyses (Fig. 2) clearly indicate that Shiraiaceae and Phaeosphaeriaceae are distinct. Evidence is also borne out by the fact the Phaeosphaeriaceae have single ascostromata , while in Shiraiaceae, ascostromata have multiple ascomata. Moreover, Shiraiaceae produces a high quantity of hypocrellins and no such metabolites, secreted by Phaeosphaeriaceae, were reported as far as we know . In this study, the third genus (i.e. Rubroshiraia) is introduced to the family and produces hypocrellins. The endophytic strains in the phylogenetic tree in Figure  (2) probably can be named as new genera, once the types are selected. Thus, currently three genera are placed in Shiraiaceae.
Type species. R. bambusae D.Q. Dai & K.D. Hyde. Notes. The hypocrellin-producing fungus R. bambusae is a well-known taxon used in Chinese traditional medicine which is called "Zhuhongjun" or "Zhuxiaorouzhuojun" in Chinese. However, without molecular data, it was wrongly named as H. bambusae (Liu 1978).
Hypocrella bambusae was combined by Saccardo (1878), based on its linear asci and filiform ascospores. Index Fungorum (2019) lists its basionym as Hypocrea bambusae Berk. & Broome, which was collected on the inflorescences of bamboo in Sir Lanka and had linear asci and filiform ascospores (Berkeley and Broome 1875). Liu (1978) recorded a well-known Chinese medicinal ascomycete, producing 0.7-1.5 mm diam., hemispheric and reddish stromata with multi-locules, cylindrical asci and filiform ascospores which are spirally arranged and more than 250 µm long on bamboo culms. Liu (1978) identified this fungus as H. bambusae, probably based on its cylindrical asci and filiform ascospores. In addition, species of Hypocrella usually produce perithecial ascomata (Saccardo 1878). To our knowledge, no fungal records or herbal medicine like that described in Liu (1978) occur in Sir Lanka. Moreover, based on the examination of type material of Hypocrea bambusae, it has smaller (0.1 cm vs. 0.7-1.5 mm in diam.) and black stromata, unitunicate asci and ascospores are in a single fascicle but not significantly helically coiled (Fig. 7). Hence, we conclude that Liu (1978) made a wrong identification.
New collections of "Zhuhongjun" were collected and sequenced. The phylogenetic analyses showed it belongs to Shiraiaceae and is separate from Shiraia with high bootstrap support (100/1.00 MLBS/BSPP) (Fig. 2). Grandigallia has not been included in the phylogenetic tree as it is lacking gene sequences in the GenBank (retrieved date: 13 May 2019). However, Grandigallia can be morphologically distinguished from the new taxon in having black ascostromata and muriform ascospores (Barr 1987;). Thus, this fungus is introduced as R. bambusae gen. et sp. nov in this study.
Rubroshiraia bambusae is often confused with S. bambusicola by Chinese traditional folk residents, probably because of the similarity of their ascostromata, parasitism on bamboo host and similar efficacy of medical treatment. However, it differs from S. bambusicola by its smaller sized ascostromata (0.7-1.2 cm long × 0.7-1 cm wide vs. 1-6 cm long × 1-4 cm wide) and distinct ascospores (filiform ascospores vs. fusiform and muriform ones). Both of the above species can produce the metabolites hypocrellin A and B, whereas R. bambusae contains almost double the content compared to S. bambusicola (Fig. 4).  Since the familial placement of H. bambusae is controversial in different studies (Berkeley and Broome 1875, Saccardo 1878, Liu 1978, we re-studied the isotype. Based on morphology, we conclude that it has unitunicate asci thus related to Sordariomycetes. Description. Parasitic on living inflorescence of bamboo. Sexual morph: Stromata around 0.14 cm diam., 0.06 cm high, solitary, superficial, subglobose, fleshy to coriaceous, black, with around 20 perithecia lining the periphery, with ostioles slightly raised above stroma surface. Stromatic tissue thick, brown to dark brown. Perithecia in vertical section around 100 µm diam., 200 µm high, pyriform, immersed in the periphery layer of stromata. Asci more than 220 µm long, 5-6 µm diam., 8-spored, unitunicate, cylindrical, with a glassy refractive cap around 3 µm from apex to base. Ascospores around 180 µm long, 1-1.5 µm diam., in a single fascicle but not significantly helically coiled, filiform, hyaline, with 9-10 transverse septa, with rounded ends, smooth-walled. Asexual morph: Undetermined. Notes. This taxon has typical morphology of the Clavicipitaceae, which is pyriform perithecia with a gradually tapering upper part and cylindrical asci with a glassy refractive cap. New collections are required and need to be sequenced to clarify its placement.

Discussion
Members of the family Shiraiaceae are distributed from Asia to South America but so far reported only from three countries, viz. China, Japan and Venezuela (Barr et al. 1987;Liu et al. 2013). The family comprises three genera, i.e. Grandigallia, Rubroshiraia and Shiraia wherein the former genus is lacking DNA sequences and, thus in here, we did not include it in the molecular analyses (Figs 2 and 3). These genera show the typical characters of Shiraiaceae, viz. conspicuous large, tuberculate, fleshy and multi-loculate ascostromata producing bitunicate asci. Shiraia bambusicola has various types of ascostromata, such as subglobose to tuberculate with white to pinkish colours (Fig. 6). However, the phylogenetic analysis shows these specimens with different types of ascostromata belong to same species (Figs 2 and 3). Thus, we assume that the different shapes of ascostromata are because of the host and different environment conditions. Stromatal methanol extracts of Rubroshiraia and Shiraia contain Hypocrellins (Fig. 4). However, so far no extracts have been reported from Grandigallia. Fresh material of Grandigallia is essential to determine the metabolites. Rubroshiraia has darker reddish ascostromata compared with Shiraia, probably because its stromatal methanol extracts contain larger quantity of hypocrellins. Some endophytes, named as Shiraialike fungi, are known to produce hypocrellins on media. They were isolated from different parts of bamboo, such as seeds, nods and internodes (Lu et al. 2004;Mora-kotkarn et al. 2008;Liang et al. 2009;Cai et al. 2011;Zhang et al. 2014). Other Shiraia-like endophytes, isolated from the rhizome of Gastrodia, leaves of Huperzia serrata and from Triticum aestivum, phylogenetically cluster within the former group (Fig. 3). However, no hypocrellins were produced from their mycelium (Zhu et al. 2010;Wang et al. 2011Wang et al. , 2016. The bamboo tissue may be providing the needful substances for fungi to produce hypocrellins. The endophytic Shiraia-like taxa (Fig. 3) appear as a distinct genus in Shiraiaceae. The nomination will be made once the type material is available. Shiraia bambusicola has been used as a Chinese traditional folk-medicine, in curing rheumatoid arthritis, infantile convulsion and pertussis etc. for more than 400 years, because of its stromatal metabolites (Huang et al. 2001;Shen et al. 2002). Japanese scientists first obtained three perylenequinones from air-dried ascostromata of S. bambusicola and named them as hypocrellin A, B and C (Kishi et al. 1991). However, hypocrellin A was originally discovered by Wan and Chen (1981) from a different fungus on bamboo which was called as "Zhuhongjun" in Chinese and was erroneously identified as H. bambusae (Liu 1978). Later the fourth hypocrellin analogue (hypocrellin D) was named by Fang et al. (2006). Therefore, in total, four hypocrellins have so far been named. Hypocrellins are types of biologically active compounds and naturally occurring perylenequinones with photodynamic activity (Wan and Chen 1981;Kishi et al. 1991;Chowdhury et al. 2002;Liang et al. 2009;Liu et al. 2013). These secondary metabolites have gained much attention owing to their light-induced anti-tumour, anti-fungal and anti-viral activities (Wan and Chen 1981;Liang et al. 2009;Li et al. 2000a, b). In clinical trials, hypocrellin shows promising treatment for various skin diseases, such as skin cancer and white lesions of the vulva (Wan and Chen 1981;Li et al. 2000b). In China, a costly medicinal unguent named Bamboo Parasitic Fungus Ointment is made of hypocrellin B (Dai et al. 2018). Interestingly, it was proved that hypocrellin has bactericidal activities which inhibit various bacteria, such as Bacillus subtilis Ehrenberg and Micrococcus luteus Schroeter (Chen et al. 2010). In addition, hypocrellin A has an antiviral activity against human immunodeficiency virus (HIV-1) (Hudson et al. 1994) and is promising as a new-fashioned photoelectric conversion material (Li et al. 2000a).
Hypocrellin has wide application prospects, but it was earlier only found existing in ascostromata of S. bambusicola and "Zhuhongjun" (R. bambusae in this paper) (Wan and Chen 1981;Kishi et al. 1991). For gaining a high yield of Hypocrellin, scientists devoted themselves to looking for strains that can produce hypocrellin through fermentation production (Liang et al. 2009). Numerous endophytes, isolated from bamboo tissue such as culms, leaves, nodes and seeds, were published (Lu et al. 2004;Morakotkarn et al. 2007Morakotkarn et al. , 2008Liang et al. 2009;Cai et al. 2011;Shen et al. 2012Shen et al. , 2014Zhang et al. 2014), several of which had the potential for hypocrellin production (Lu et al. 2004;Morakotkarn et al. 2008;Liang et al. 2009;Zhang et al. 2014;Tong et al. 2017). However, the strains with promising industrial fermentation were identified as Shiraia sp. based on the blast search in GenBank by ITS sequences. More endophytes producing biologically active compounds, such as huperzine, isolated from the plant Huperzia serrata (Thunb. ex Murray) Trev., were also named as Shiraia sp. (Wang et al. 2011(Wang et al. , 2016Zhu et al. 2010). These strains usually have around 80%-90% ITS similarity with S. bambusicola, which also shows that they are phylogenetically close with members of Shiraiaceae. In this study, these endophytes are placed in Shiraiaceae, based on the phylogenetic analyses (Fig. 3).
According to Deng et al. (2017), polyketide synthase (SbaPKS) is involved in hypocrellin biosynthesis, based on the methods of CRISPR/Cas9 genome editing. It provides evidence for decoding the hypocrellin pathway (Deng et al. 2017). This pathway has the potential for producing high quality hypocrellins.