Morpho-molecular diversity of Linocarpaceae (Chaetosphaeriales): Claviformispora gen. nov. from decaying branches of Phyllostachys heteroclada

Abstract In this paper, Claviformisporagen. nov. in Linocarpaceae is introduced from Phyllostachys heteroclada in Sichuan Province, China. The new genus is characterised by its distinct morphological characters, such as ostiole with periphyses, asci with a thick doughnut-shaped, J- apical ring and clavate ascospore without septum-like band and appendage. Maximum Likelihood and Bayesian Inference phylogenetic analyses, based on DNA sequence data from ITS, LSU, SSU and TEF-1α regions, provide further evidence that the fungus is a distinct genus within this family. The new genus is compared with similar genera, such as Linocarpon and Neolinocarpon. Descriptions, illustrations and notes are provided for the new taxon.

The present research is a part of our investigations on the taxonomic and phylogenetic circumscriptions of pathogenic and saprobic micro-fungi associated with bamboo in Sichuan Province, China. In this paper, we introduce a new genus Claviformispora in Linocarpaceae, typified by C. phyllostachydis from Phyllostachys heteroclada Oliv., 1894 (Poaceae). The morphological differences and analyses of a combined ITS, LSU, SSU and TEF-1α sequence dataset support the validity of the new genus and its placement in Linocarpaceae. The new genus is compared with other genera in the family. The comprehensive descriptions and micrographs of new taxa are provided.

Specimen collection and morphological study
Bamboo materials were collected from Ya'an City, China. Single ascospore isolations were carried out following the method described by Chomnunti et al. (2014) and the germinating spores were transferred to PDA, incubated at 25 °C in the dark and cultural characteristics were determined. Ascomata were observed and photographed using a dissecting microscope NVT-GG (Shanghai Advanced Photoelectric Technology Co. Ltd, China) matched to a VS-800C micro-digital camera (Shenzhen Weishen Times Technology Co. Ltd., China). The anatomical details were visualised using Nikon ECLIPSE Ni compound microscope fitted to a Canon 600D digital camera and an OPTEC BK-DM320 microscope matched to a VS-800C micro-digital camera (Shenzhen Weishen Times Technology Co. Ltd., China). Iodine reaction of the ascus wall was tested in Melzer's reagent (MLZ). Lactate cotton blue reagent was used to observe the number of septa. The gelatinous appendage was observed in Black Indian ink. Type specimens were deposited at the Herbarium of Sichuan Agricultural University, Chengdu, China (SICAU) and Mae Fah Luang University Herbarium (MFLU). The ex-type living cultures are deposited at the Culture Collection in Sichuan Agricultural University (SICAUCC) and the Culture Collection at Mae Fah Luang University (MFLUCC). Index Fungorum numbers (http://www.indexfungorum.org/Names/ Names.asp) are registered and provided.

DNA extraction, PCR amplification and DNA sequencing
Total genomic DNA was extracted from mycelium that were grown on PDA at 25 °C for two weeks using a Plant Genomic DNA extraction kit (Tiangen, China) following the manufacturer's instructions. The primers pairs LR0R and LR5 (Vilgalys and Hester 1990), NS1 and NS4, ITS5 and ITS4 (White et al. 1990), EF1-983F and EF1-2218R (Rehner 2001) were used for the amplification of the partial large subunit nuclear rDNA (LSU), the partial small subunit nuclear rDNA (SSU), internal transcribed spacers (ITS) and translation elongation factor 1-alpha (TEF-1α), respectively.
Polymerase chain reaction (PCR) was performed in 25 μl final volumes containing 22 μl of Master Mix (Beijing TsingKe Biotech Co. Ltd.), 1 μl of DNA template, 1 μl of each forward and reverse primers (10 μM). The PCR thermal cycle programmes for LSU, SSU, ITS and TEF1-α gene were amplified as: initial denaturation 94 °C for 3 minutes, followed by 35 cycles of denaturation at 94 °C for 30 seconds, annealing at 55 °C for 50 seconds, elongation at 72 °C for 1 minute and final extension at 72 °C for 10 minutes. PCR products were sequenced with the above-mentioned primers at TsingKe Biological Technology Co. Ltd, Chengdu, China. The newly-generated sequences from the LSU, SSU, TEF-1α and ITS regions were deposited in GenBank (Table 1).
Notes. New species in this study is in bold. "-" means that the sequence is missing or unavailable.

Phylogenetic analyses
Taxa to be used for phylogenetic analyses were selected, based on results generated from nucleotide BLAST searches online in GenBank and recent publications (Lu et al. 2016;Konta et al. 2017;Senwanna et al. 2018;Wei et al. 2018;Lin et al. 2019). Gelasinospora tetrasperma (CBS 178.33) and Sordaria fimicola (CBS 508.50) were selected as the outgroup taxa. The sequences were downloaded from GenBank (http:// www.ncbi.nlm.nih.gov/) and the accession numbers are listed in Table 1. A combined ITS, LSU, SSU and TEF-1α sequence dataset was used to construct the phylogenetic tree. DNA alignments were performed by using MAFFT v.7.429 online service (Katoh et al. 2019) and ambiguous regions were excluded with BioEdit version 7.0.5.3 (Hall 1999). Multigene sequences were concatenated by using Mesquite software (Maddison and Maddison 2019). Maximum Likelihood (ML) and Bayesian Inference (BI) analyses were performed. The best nucleotide substitution model was determined by MrModeltest v. 2.2 (Nylander 2004). Maximum Likelihood analysis and Bayesian Inference analysis were generated by using the CIPRES Science Gateway web server (Miller 2010). RAxML-HPC2 on XSEDE (8.2.10) (Stamatakis 2014) with GTR+GAMMA substitution model with 1000 bootstrap iterations was chosen for Maximum Likelihood analysis. For BI analyses, the best-fit model GTR+I+G for ITS, LSU and SSU was selected in MrModeltest 2.2 and GTR+G for TEF. The analyses were computed with six simultaneous Markov Chain Monte Carlo (MCMC) Chains with 8,000,000 generations and a sampling frequency of 100 generations. The burn-in fraction was set to 0.25 and the run automatically ended when the average standard deviation of split frequencies reached below 0.01.
Phylogenetic trees were visualised with FigTree v.1.4.3 (Rambaut and Drummond 2016) and edited using Adobe Illustrator CS6 (Adobe Systems Inc., United States). Maximum Likelihood bootstrap values (MLBP) equal to or greater than 70% and Bayesian Posterior Probabilities (BYPP) equal to or greater than 0.95 were accepted. The finalised alignment and tree were deposited in TreeBASE (http://www.treebase. org), submission ID: 25996. The new taxa introduced follow the recommendations of Jeewon and Hyde (2016).

Phylogenetic analyses
Phylogenetic analyses of a combined dataset (ITS, LSU, SSU, TEF-1α) comprises 51 taxa within the order Chaetosphaeriales (Table 1), including 24 taxa in family Chaetosphaeriaceae, nine taxa in Helminthosphaeriaceae, ten taxa in Linocarpaceae, six taxa in Leptosporellaceae and two outgroup taxa in Sordariales. The dataset consisted of 5,849 characters including gaps (LSU = 1,571, ITS = 736, SSU = 2,522, TEF = 1,020). The best scoring tree of RAxML analysis is shown in Fig. 1, with the support values of ML and BI analyses. greater than 70% and Bayesian posterior probabilities (PP, right) equal to or greater than 0.95 are indicated at the nodes. The tree is rooted to Gelasinospora tetrasperma (CBS 178.33) and Sordaria fimicola (CBS 508.50). All sequences from ex-type strains are in bold. The newly-generated sequence is in red.
The best scoring RAxML tree with the final optimisation had a likelihood value of -26,415.700648. The matrix had 1,751 distinct alignment patterns and 64.64% in this alignment is the gaps and completely undetermined characters. Estimated base frequencies were as follows: A = 0.236065, C = 0.261532, G = 0.295313, T = 0.207091, with substitution rates AC = 1.062535, AG = 1.855434, AT = 0.940219, CG = 1.052604, CT = 4.590285, GT = 1.000000. The gamma distribution shape parameter α = 0.311923 and the Tree-Length = 2.281738. The Bayesian analysis resulted in 20,502 trees after 8,000,000 generations. The first 25% of trees (1,624 trees), which represent the burnin phase of the analyses were discarded, while the remaining 4,878 trees were used for calculating posterior probabilities. Bayesian posterior probabilities were evaluated by MCMC with a final average standard deviation of split frequencies = 0.009877.
Phylogenetic trees generated from Maximum Likelihood (ML) and Bayesian Inference analyses were similar in overall topologies. Phylogeny from the combined sequence data analysis indicates that all families were monophyletic with strong bootstrap support values (Fig. 1). Phylogenetic results show that our novel species Claviformispora phyllostachydis (SICAUCC 16-0004) belongs to family Linocarpaceae with 91% ML and 1.00 BYPP support and close to genera Neolinocarpon and Linocarpon (Fig. 1). The new genus Claviformispora constituted a distinct lineage in the family Linocarpaceae (Fig. 1). Description. Saprobic and endophytic fungi on monocotyledons and rarely dicotyledons. Sexual morph: Ascomata solitary or aggregated, superficial or immersed comprising black, dome-shaped or subglobose, slightly raised blistering areas with a central ostiole or immersed with a black shiny papilla. Peridium composed of dark brown to black cells of textura angularis. Hamathecium comprising septate paraphyses that are longer than asci, wider at the base, tapering towards the apex. Asci 8-spored, unitunicate, cylindrical, with a J-, apical ring, developing from the base and periphery of the ascomata. Ascospores parallel or spiral in asci, hyaline or pale yellowish in mass, filiform or claviform, straight or curved, unicellular with or without refringent bands, with or without polar appendages. Asexual morph: Phialophora-like spp. were found in Linocarpon appendiculatum and L. elaeidis cultures (Hyde 1992b), but no records are available for other species.

Taxonomy
Notes. Linocarpaceae was introduced as a new family to accommodate Linocarpon and Neolinocarpon species, based on morphology and phylogeny (Konta et al. 2017). Appressoria were first recorded from Neolinocarpon rachidis . The new genus Claviformispora, which is well-supported within Linocarpaceae suggests that there is a need to amend the morphological circumscriptions of the family given that the ascomata (subglobose) and ascospore (claviform) characters are so different from the other two genera. Etymology. Name reflects the claviform ascospores. Description. Saprobic on dead branches. Sexual morph: Stromata solitary or gregarious, black, erumpent. Ascomata solitary or aggregated, immersed, subglobose, slightly raised blistering areas with a central ostiole with periphyses. Peridium outer cells merging with the host tissues, composed of pale to dark brown cells of textura angularis. Hamathecium comprising hyaline, septate paraphyses, longer than asci, wider at the base, tapering towards the apex. Asci 8-spored, cylindrical to cylindric-clavate, unitunicate, short pedicellate, apically rounded, with a doughnut-shaped, refractive, J-apical ring. Ascospores overlapping uniseriate or 2-seriate, clavated with a thin pedicellate, 1-celled, hyaline, without appendage and refringent bands, smooth-walled. Asexual morph: Undetermined.
Culture characters. Ascospores germinated on PDA within 12 hours at both ends. Colonies on PDA reaching 5 cm diameter after 7 days at 25 °C, white to grey with strong radiations outwards on forward side. Colonies became dark brown and black on the reverse after a long time of cultivation. The hyphae are septate, branched, smooth.

Discussion
This study establishes a new genus and also provides further insights into the phylogeny of members associated with Linocarpaceae. Morphologically-based examinations of Claviformispora (as discussed above) clearly show that the morphological circumscriptions (familial concept) of species should be broadened and possibly indicate that this family is much more diverse than expected. Our collection can be clearly distinguished from other groups of similar fungi in Linocarpaceae with its interesting ascospore morphology. In addition, we also noted some peculiarities in the DNA sequences we analysed. A comparison of ITS sequences, based on BLAST reveals 34%, 26% and 30% base pair differences with L. cocois , N. arengae  and N. rachidis (MFLUCC 15-0814a), respectively. There are more than 9% and 5% sequence differences with the three taxa when the LSU and SSU rDNA sequences were compared respectively. Following the guidelines recommended by Jeewon and Hyde (2016), there are therefore sufficient grounds to establish a new species at the genus rank. Species of Linocarpaceae have been found on Arecaceae, Poaceae, Euphorbiaceae, Zingiberaceae, Pandanaceae, Fagaceae, Fabaceae and Smilacaceae, including Arenga, Attalea, Calamus, Trachycarpus, Acrocomia, Archontophoenix, Cocos, Daemonorops, Licuala, Livistona, Plectocomia, Phoenix, Raphia, Sabal, Mauritia, Nypa, Elaeis, Pinanga, Eugeissona, Pennisetum, Gramineae, Stipa, unidentified bamboo, Hevea, Manihot, Alpinia, Pandanus, Quenrcus, Cajanus andSmilax (Hyde 1988, 1992a, b;Dulymamode et al. 1998;Hyde et al. 1998;Hyde and Alias 1999;Thongkantha et al. 2003;Cai et al. 2004;Bhilabutra et al. 2006;Vitoria et al. 2013;Konta et al. 2017;Senwanna et al. 2018). More than 50% of the species were recorded from hosts of the Arecaceae. Species in Linocarpaceae are mostly saprobic, except Linocarpon palmetto which was discovered as a pathogen of Sabal palmetto in Florida (Barr 1978). Four species in Linocarpaceae from Poaceae have been reported so far, including Neolinocarpon penniseti on Pennisetum purpureum (Bhilabutra et al. 2006), Linocarpon williamsii on Gramineae sp. (Hansford 1954), L. stipae on Stipa sp. (Hansford 1954) and L. bambusicola on unidentified bamboo submerged in a river (Cai et al. 2004).
Phyllostachys heteroclada, mainly a food source and use as a material in the weaving industry, is distributed along the Yellow River Valley and the southern Provinces in China. It is common in the mountainous areas of Sichuan Province with distribution up to 1,500 m above sea level (Yi 1997;Yi et al. 2008). There is a large area of pure forest in Yibin, Leshan and Ya'an Cities and sporadic distribution in other areas. According to preliminary statistics, bambusicolous fungi from seven orders (excluding fungi referred to as Sordariomycetes incertae sedis) have been recorded on P. heteroclada, including Hypocreales, Ostropales, Pleosporales, Phyllachorales, Pucciniales, Ustilaginales and Xylariales, of which Pleosporales is the largest one. Most bambusicolous fungi in China were recorded with inadequate morphological descriptions or molecular data. The early known fungi on P. heteroclada are documented as Aciculosporium take, Ellisembia pseudoseptata, Fusarium oxysporum, F. semitectum, Phyllachora gracilis, Ph. orbicular, Shiraia bambusicola, Stereostratum corticioides and Ustilago shiraiana (Zhou et al. 2001;Xu et al. 2006). In recent years, some new records and taxa, viz. Bambusicola subthailandica, B. sichuanensis, Neostagonosporella sichuanensis, Parakarstenia phyllostachydis, Phyllachora heterocladae, Podonectria sichuanensis, Arthrinium yunnanum and A. phyllostachium have been reported (Yang et al. 2019a, b, c, d, e, f ). Here, we introduce a new genus in order Chaetosphaeriales, which is a contribution to fungal diversity on P. heteroclada.