Introduction

Xylariales is a large order of perithecial ascomycetes with eight-spored unitunicate asci, usually with a J+ apical ring and ascospores with a prominent germ pore or germ slit and is accommodated in the subclass Xylariomycetidae (Smith et al. 2003; Senanayake et al. 2015; Maharachchikumbura et al. 2016). Asexual morphs of the Xylariales are usually hyphomycetous, with holoblastic conidiogenesis (Maharachchikumbura et al. 2015, 2016). Presently, there are 22 accepted families in the Xylariales, viz. Amphisphaeriaceae, Apiosporaceae, Bartaliniaceae, Beltraniaceae, Cainiaceae, Clypeosphaeriaceae, Coniocessiaceae, Diatrypaceae, Hyponectriaceae, Iodosphaeriaceae, Lopadostomaceae, Melogrammataceae, Microdochiaceae, Myelospermataceae, Pestalotiopsidaceae, Phlogicylindriaceae, Pseudomassariaceae, Requienellaceae, Robillardaceae, Sporocadaceae, Vialaeaceae and Xylariaceae (Maharachchikumbura et al. 2015, 2016).

A fungal tribe named Beltranieae Sacc. was established by Saccardo (1886) to accommodate a single genus Beltrania Penzig. Nannizzi (1934) introduced Beltraniaceae Nann. to accommodate Beltrania and some similar genera, and the tribe Beltranieae was treated as a synonym of this family. Crous et al. (2015b) emended the family Beltraniaceae and accepted Beltrania, Beltraniella Subram., Beltraniopsis Bat. & J.L. Bezerra, Parapleurotheciopsis P.M. Kirk and Pseudobeltrania Henn. They provided DNA sequence data to support the family. Crous et al. (2015b) proposed that three more genera, Beltraniomyces Manohar., D.K. Agarwal & Rao, Porobeltraniella Gusmão and Subramaniomyces Varghese & V.G. Rao, should be accepted in this family. Due to the lack of reliable strains and sequence data, they could not confirm the familial relationships of these genera within the Beltraniaceae. Maharachchikumbura et al. (2016) accepted the genus Subramaniomyces within the family Beltraniaceae based on phylogenetic analysis. Rajeshkumar et al. (2016a) accepted the genera Hemibeltrania and Porobeltraniella and preliminarily confirmed the monophyly of all of the recognised genera based on phylogenetic analysis.

The asexual morphs in Beltraniaceae are hyphomycetous (Seifert et al. 2011; Crous et al. 2015b). Stromata are usually present. Setae are present or absent, branched or unbranched and usually with a radially lobed basal cell. The conidiophores are branched or unbranched, arising from the base of setae or separate, sometimes arising from radially lobed basal cells. The conidiogenous cells are monoblastic or polyblastic, sympodial, integrated or discrete and denticulate. Separating cells are present or absent, oval to subglobose, also with one to several denticles. Conidia are biconic, lageniform to navicular, hyaline to red-brown, generally with a lightly pigmented transverse band at the widest part of the conidium, rounded or 1-denticulate or rostrate at the base and spicate or apiculate or truncate at the apex (Crous et al. 2015b).

During a survey of hyphomycetes in Karst areas of Thailand, a Beltrania-like species was collected. It was shown to belong to a new genus in Beltraniaceae based on morphology and analyses of ITS and LSU sequence data. The natural classification of this new taxon is determined based on phylogenetic analysis and morphology.

The new taxon is morphologically similar to Beltrania and some similar genera, but its conidiophores and conidiogenous cells differ from all previously described genera of this group. We propose a new genus to accommodate the new fungus which is introduced here.

Materials and methods

Collection and isolation of fungi

Dead materials (stem, wood and leaves) from a variety of plants were randomly collected during July to August 2015 from Karst areas at Khao Lom Muak (11°47′3.96″–11.24″N, 99°48′49.13″–49′0.63″E), Prachuap Khiri Khan in Thailand. Samples were taken to the laboratory in zip-lock plastic bags for examination. The specimens were incubated in sterile moist chambers and examined using a Motic SMZ-168 series microscope (Speed Fair Co., China). Fungi were removed with a needle and placed in a drop of distilled water on a slide for morphological study. Photomicrographs of fungal structures were captured using a Nikon Eclipse 80i compound microscope (Nikon Co., Japan) with a Canon 450D digital camera (Canon Co., Japan). All measurements were made by the Tarosoft® Image FrameWork program (Tarosoft, Thailand). Photo-plates were made with Adobe Photoshop CS6 Extended version 13.0.1 (Adobe Systems, USA). Isolation onto potato dextrose agar (PDA) or malt extract agar (MEA) was performed by the single spore isolation method (Chomnunti et al. 2014; Dai et al. 2017). The herbarium material is deposited in the herbarium of Mae Fah Luang University (MFLU), Chiang Rai, Thailand and the herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (HKAS), Kunming, China. Cultures are deposited at the Mae Fah Luang University Culture Collection (MFLUCC), Chiang Rai, Thailand and Kunming Institute of Botany, Chinese Academy of Sciences (KUMCC), Kunming, China. Faces of Fungi and Index Fungorum numbers are registered (Jayasiri et al. 2015; Index Fungorum 2016). Colours and colour codes were determined according to Kornerup and Wanscher (1978).

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from fungal mycelium grown on PDA or MEA at room temperature with the Fungal gDNA Kit (BioMIGA, USA), according to the manufacturer’s instructions. The internal transcribed spacer region of ribosomal DNA (ITS) and large subunit nuclear ribosomal DNA (LSU) genes were amplified via polymerase chain reaction (PCR) using the following primers: ITS5 and ITS4 (White et al. 1990) for ITS and LROR and LR5 (Vilgalys and Hester 1990) for LSU. The PCR products were sequenced with the same primers.

Phylogenetic analyses

Original sequences were checked using BioEdit version 7.0.5.3 (Hall 1999) and most reference sequences originated from previous publications, viz. Crous and Groenewald (2013), Crous et al. (2014, 2015a, b) and Maharachchikumbura et al. (2015). The remaining homologous sequences were obtained by BLAST searches (Altschul et al. 1990) from GenBank. All sequences used in this study are listed in Table 1. Alignments for each locus were done in MAFFT v7.212 (Katoh and Standley 2013) and manually verified in MEGA 6.06 (Tamura et al. 2013). Conserved blocks were selected from the initial alignments with Gblocks 0.91b (Castresana 2000). The interleaved NEXUS files were formatted with PAUP*4.0b10 (Swofford 2002) and manually formatted for Bayesian inference analyses. Bayesian inference (BI), maximum parsimony (MP) and maximum likelihood (ML) were used in this study for phylogenetic analyses. For Bayesian inference analysis, the best model of evolution was determined using MrModeltest v2 (Nylander 2004). Bayesian inference analysis was done with MrBayes v3.2.5 (Ronquist et al. 2012). Maximum parsimony analysis was performed in PAUP*4.0b10 (Swofford 2002). Maximum likelihood analysis was performed in raxmlGUI v1.3.1 (Silvestro and Michalak 2012). Phylogenetic trees were drawn with TreeView 1.6.6 (Page 1996).

Table 1 GenBank accession numbers of isolates used in this study
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Results

Molecular phylogeny

The aligned sequence matrix comprises LSU (848 bp) and ITS (633 bp) sequence data for 40 taxa and one outgroup taxon for a total of 1481 characters, of which 267 were parsimony-informative, 100 were parsimony-uninformative and 1114 characters were constant. The result of maximum likelihood (ML) analysis based on combined LSU and ITS sequence data consisting of five families (Amphisphaeriaceae, Apiosporaceae, Beltraniaceae, Pestalotiopsidaceae and Robillardaceae) within the order Xylariales is shown in Fig. 1.

Fig. 1
figure 1

Phylogenetic tree generated from maximum likelihood (ML) analysis based on combined LSU and ITS sequence data for some selected families within the order Xylariales. Bootstrap support values for maximum likelihood (ML) and maximum parsimony (MP) greater than 50% and Bayesian posterior probabilities greater than 0.8 are indicated above or below the nodes as MLBS/MPBS/PP. The ex-type strains are in bold and the new isolate is in bold and red. The tree is rooted with Anthostomella leucospermi (CBS 110126)

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In the present study, we found that the strain of Subsessila turbinata (MFLUCC 15-0831) grouped together with Hemibeltrania sp. (CL12WA) and Hemibeltrania cinnamomi (NFCCI 3695 and NFCCI 3997) with 68% ML bootstrap support, 63% MP bootstrap support and 99% Bayesian posterior probabilities within the family Beltraniaceae (Fig. 1). The genera Hemibeltrania, Porobeltraniella, Pseudobeltrania and Subsessila clustered together with 63% ML bootstrap support, 89% MP bootstrap support and 100% Bayesian posterior probabilities.

Taxonomy

Subsessila C.G. Lin & K.D. Hyde, gen. nov.

Index Fungorum number: IF552504; Facesoffungi number: FoF 02613

Etymology: In reference to the conidiophores which are mostly absent or reduced to conidiogenous cells.

Type species: Subsessila turbinata C.G. Lin & K.D. Hyde

Saprobic on plant host. Asexual morph: Colonies on plant substrate effuse, pale brown, hairy, velvety. Mycelium partly superficial and partly immersed. Stroma absent. Setae numerous, erect, arising from radially lobed basal cells, straight or slightly flexuous, unbranched, 2–4-septate, thick-walled, verrucose, pale to dark brown, swollen at the base, tapering towards the apex. Conidiophores mostly absent or reduced to conidiogenous cells; when present, arising from the basal cells of setae, simple, aseptate, subcylindrical, pale- to mid-brown, smooth. Conidiogenous cells polyblastic, discrete, determinate, ampulliform, doliiform, hyaline. Separating cells absent. Conidia aggregated, acrogenous, simple, dry, straight, smooth, thin-walled, turbinate to clavate, rostrate at proximal end, rounded at distal end, hyaline. Sexual morph: Undetermined.

Subsessila turbinata C.G. Lin & K.D. Hyde, sp. nov. (Fig. 2)

Fig. 2
figure 2

Subsessila turbinata (holotype MFLU 15-3271). a Leaf material. b, c Conidiophores on the host surface. d Conidiophores, seta and conidia. e Short conidiophores with conidiogenous cell. f, g Conidiogenous cell and conidia. h–j Conidia. k Germinating conidium. l, m Colonies on MEA culture, l from above, m from below. Scale bars: b = 500 μm, c = 200 μm, d = 50 μm, e–k = 10 μm

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Index Fungorum number: IF552503; Facesoffungi number: FoF 02614

Etymology: In reference to the turbinate conidia.

Holotype: MFLU 15-3271

Saprobic on plant host. Asexual morph: Colonies on plant substrate effuse, pale brown (6C2), hairy, velvety. Mycelium partly superficial and partly immersed. Stroma absent. Setae numerous, erect, arising from radially lobed basal cells, straight or slightly flexuous, unbranched, 2–4-septate, thick-walled, verrucose, medium to dark brown (6C4 to 6F8), 50–280 μm long, swollen at the base and 4.5–13 μm wide, 2–7 μm wide just above the swollen base, tapering to a pointed apex. Conidiophores mostly absent and reduced to conidiogenous cells; if present, arising from basal cells of setae, simple, aseptate, subcylindrical, pale- to mid-brown (6C2 to 6C4), up to 6.5 μm long, 3–7.5 μm wide. Conidiogenous cells polyblastic, discrete, determinate, ampulliform, doliiform, hyaline, smooth, 4.5–6.5 μm (\( \overline{x} \) = 5.5 μm, n = 30) long, 4–6 μm (\( \overline{x} \) = 5.1 μm, n = 30) wide in the broadest part. Separating cells absent. Conidia aggregated, acrogenous, simple, dry, straight, smooth, thin-walled, turbinate to clavate, rostrate to pointed at proximal end, broadly rounded at distal end, hyaline, 14.5–20.5 μm (\( \overline{x} \) = 17.70 μm, n = 50) long, 4.5–6.5 μm (\( \overline{x} \) = 5.50 μm, n = 50) wide in the broadest part. Sexual morph: Undetermined.

Culture characteristics: Conidia germinating on MEA within 12 h. Colonies on MEA effuse, greyish white (−B1) from above, greyish yellow (2B5) from below, reaching a diam. of 2–3 cm in 20 days at 25 °C.

Material examined: THAILAND, Prachuap Khiri Khan, Khao Lom Muak, 11°47′3.96″–11.24″N, 99°48′49.13″–49′0.63″E, on unidentified decaying leaf, 29 July 2015, Chuan-Gen Lin, KLM 8-1 (MFLU 15-3271, holotype; HKAS 96229, isotype), ex-type living culture MFLUCC 15-0831, KUMCC 16-0126.

Discussion

In the tree generated from the maximum likelihood (ML) analysis based on combined ITS and LSU sequence data for the order Xylariales, Subsessila turbinata (MFLUCC 15-0831) grouped together with Hemibeltrania sp. (CL12WA) and Hemibeltrania cinnamomi (NFCCI 3695 and NFCCI 3997) with 100% ML bootstrap support, 99% MP bootstrap support and 100% Bayesian posterior probabilities within the family Beltraniaceae (Fig. 1). Subsessila differs from Hemibeltrania and Pseudobeltrania by the absence of distinct conidiophores, which, whenever present, are very short and arise from the basal cells of setae; terminal, ampulliform, doliiform, hyaline conidiogenous cells and turbinate to clavate, subhyaline, smooth conidia which are rostrate to pointed at the proximal end and broadly rounded at the rostrate distal end. In addition, Subsessila has numerous, unbranched setae arising from radially lobed basal cells. Stroma and separating cells are not observed in these three genera.

Morphologically, Subsessila is similar to several genera within the family Beltraniaceae, viz. Beltrania, Beltraniella, Beltraniopsis and Porobeltraniella, in having dark setae and conidiophores arising from radially lobed basal cells (Fig. 2). However, Beltrania, Beltraniella, Beltraniopsis and Porobeltraniella have distinct, swollen separating cells, and conidia of these four genera are turbinate or biconic, with a hyaline transverse band or several equatorial hyaline pores, whereas separating cells are not present in Subsessila and the conidia are turbinate to clavate, with a rostrate to pointed proximal end and rounded distal end. The most distinguishable characters that separate Subsessila from Beltrania and other similar genera are the absence of distinct conidiophores and conidia without a hyaline transverse band in the new genus.

With the above combination of morphological features and phylogenetic analysis, we place the new genus Subsessila within the family Beltraniaceae.

Presently, 14 genera, including our new genus, have some similar characters that are present in the “Beltrania complex”, viz. Beltrania Penzig, Beltraniella Subram., Beltraniomyces Manohar., D.K. Agarwal & Rao, Beltraniopsis Bat. & J.L. Bezerra, Beltramono Dubey, Pandey & Manohar., Hemibeltrania Piroz., Kiliophora Kuthub. & Nawawi, Maxibeltrania Rambelli, Parabeltrania Rambelli, Porobeltraniella Gusmão, Pseudobeltrania Henn., Rhombostilbella Zimm., Scolecobeltrania Iturr., R.F. Castañeda & R. Fernández and Subsessila. A synopsis of Beltrania and similar genera is provided in Table 2. These genera have unbranched or branched conidiophores and/or setae arising from radially lobed basal cells, with or without swollen separating cells, and biconic conidia with or without a hyaline equatorial or sub- or supraequatorial band. Presently, six of them, viz. Beltrania, Beltraniella, Beltraniopsis, Hemibeltrania, Porobeltraniella and Pseudobeltrania, are accepted in the family Beltraniaceae.

Table 2 Synopsis of Beltrania and some similar genera [updated from Manoharachary et al. (2003) and Dubey et al. (2011)]
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Kendrick (1980) proposed that the genera in the Beltrania complex must possess any three of the five following features: (1) dark setae; (2) setae or conidiophores with radially lobed bases; (3) swollen separating cells; (4) biconic conidia; (5) conidia with a hyaline equatorial band. There are nine genera showing Beltrania-like morphological features (Table 3), viz. Beltrania, Beltraniella, Beltraniopsis, Beltramono, Maxibeltrania, Parabeltrania, Porobeltraniella, Pseudobeltrania and Scolecobeltrania. Within this, Beltraniomyces, Hemibeltrania, Kiliophora, Rhombostilbella and Subsessila show some differences in that Beltraniomyces and Subsessila have only two of those characteristics, whereas Hemibeltrania, Kiliophora and Rhombostilbella have only one of those characteristics.

Table 3 Salient features of Beltrania and some similar genera [updated from Kendrick (1980)]
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