Arthrinium bambusicola (Fungi, Sordariomycetes), a new species from Schizostachyum brachycladum in northern Thailand

Abstract Background Species of the fungal genus Arthrinium (Sordariomycetes, Amphisphaeriales, Apiosporaceae) are often found on bamboo in Asia. They are endophytes, saprobes and important plant pathogens. The genus Arthrinium currently contains 92 species and is widely distributed in North and South America, Europe, Africa, Asia and Oceania. New information In this study, a new species, Arthrinium bambusicola sp. nov., is described and illustrated. The new taxon is characterised by oval to broadly or irregularly round, medium brown, multi-guttulate to roughened, granular conidia, with finely pale slits in the outer edges. Arthrinium bambusicola can be distinguished from the closest related species A. gutiae by its conidial characteristics. Phylogenetic analyses of a four-locus dataset (ITS, LSU, TEF1, TUB2) confirm that A. bambusicola is a distinct new species.


Introduction
The genus Arthrinium, with A. caricicola as type species, was established by Schmidt and Kunze (Kunze 1817). Species of Arthrinium are endophytes, saprobes and important plant pathogens of various hosts, particularly grasses and bamboo (Agut and Calvo 2004, Rashmi et al. 2019. The sexual morph is characterised by black, linear, fusiform ascostromata with a long, slit-like opening at the apex. The ascomata are globose to subglobose, with flattened bases and brown to blackish, with or without setae , Pintos et al. 2019).
Species of Arthrinium produce both hyphomycetous and coelomycetous asexual morphs. The hyphomycetous morph is characterised by septate conidiophores, arising from basal cells or that are reduced to conidiogenous cells. Conidiogenous cells are holoblastic, monoblastic or polyblastic and are hyaline to pale brown, smooth or finely roughened, doliiform, ampulliform or subcylindrical and conidia are dark brown, brown to pale olivaceous and of various shapes (Hyde et al. 2016). The coelomycetous morph is immersed, black, globose to subglobose, septate, hyphoid conidiomata and hyaline to pale brown conidiophores arising from basal cells or that are reduced to conidiogenous cells. The conidiogenous cells are subhyaline to pale brown, smooth-walled or verrucose, holoblastic, monoblastic or polyblastic and cylindrical. The conidia are dark brown, smooth, globose to subglobose, with or without a germ slit or truncate scar at the base (Senanayake et al. 2015, Jiang et al. 2018. The presence of both hyphomycetous and coelomycetous asexual morphs has complicated the taxonomy of Arthrinium . There are 92 species epithets for Arthrinium in Index Fungorum (2020). A total of 63 species have been introduced, based on the combination of morphological and molecular phylogenetic data , Jiang et al. 2018, Pintos et al. 2019, Yan et al. 2019. In this study, we propose a new species, based on morphological study and comparison with other species, in combination with phylogenetic analyses of a concatenated dataset of ITS, LSU, TEF1 and TUB2 sequences.

Sample collection and isolation
Fresh samples of dead culms of Schizostachyum brachycladum (Poales, Poaceae and Bambusoideae) were collected at the campus of Mae Fah Luang University, Chiang Rai, Thailand on 7 May 2019. Single-spore isolation was performed as in Chomnunti et al. (2014). The holotype is deposited at the herbarium of Mae Fah Luang University, Chiang Rai, Thailand (MFLU) and the ex-type living culture is preserved at the Mae Fah Luang University Culture Collection (MFLUCC). Facesoffungi and Index Fungorum numbers for the new taxon were obtained (Jayasiri et al. 2015, Index Fungorum 2020.

Morphological examination
Conidiomata present on the surface of the host were observed using a stereomicroscope (Motic SMZ-171, Wetzlar, Germany). Sections of conidiomata were taken and mounted in water on a microscope slide to observe fungal characters. Photographs were taken using a Nikon ECLIPSE Ni-U compound microscope connected with a Nikon camera series DS-Ri2. Morphological structures (conidiophores, conidiogenous cells, conidia) were measured by Image Frame Work software v. 0.9.7. Adobe Photoshop CC 2019 was used for editing the photographic plate. Colonies were described, based on the colour charts of Rayner (1970).

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from fresh mycelia obtained from living cultures that were grown on potato dextrose agar (PDA) for 15 days at room temperature, using the EZgene Fungal gDNA Kit (GD2416, Biomiga, San Diego, California, USA) following the manufacturer's instructions. PCR amplification was done for the internal transcribed spacer region (ITS), the large subunit of the ribosomal RNA gene (LSU), translation elongation factor 1-alpha (TEF1) and beta-tubulin (TUB2). The following primers were used: ITS5 and ITS4 for ITS (White et al. 1990); LR0R and LR5 for LSU (Vilgalys andHester 1990, Hopple 1994); EF1-728F and EF-2 for TEF1 (O'Donnell et al. 1998, Carbone andKohn 1999).
PCR amplification was done in 50-μl volumes consisting of 2 μl of DNA template, 2 μl of each 10 μM forward and reverse primers, 25 μl of 2 ×Taq PCR Master Mix and 19 μl of deionised water. Cycling conditions were as follows: for ITS: initial denaturation at 94°C for 5 min, then 35 cycles of denaturation at 94°C for 45 s, annealing at 52°C for 50 s and extension at 72°C for 1 min; and final extension at 72°C for 10 min. For LSU: initial denaturation at 94°C for 5 min, then 35 cycles of denaturation at 94°C for 45 s, annealing at 52°C for 50 s and extension at 72°C for 1 min; and final extension at 72°C for 10 min.
Lastly, for TEF1: initial denaturation at 94°C for 5 min; then 35 cycles of denaturation at 94°C for 1 min, annealing at 56°C for 1 min and extension at 72°C for 90 s; and final extension at 72°C for 10 min.
PCR products were checked in 1% agarose gels and sent to Sangon Biotech (Shanghai) Co. Ltd, China for sequencing, using the same primers.

Phylogenetic analyses
Raw sequence reads were combined using BioEdit v. 7.0.5.3 (Hall 1999) and subjected to BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to find closely-related taxa. To confirm the phylogenetic position of our taxon, sequences of four loci (ITS, LSU, TEF1 and TUB2) were downloaded from NCBI GenBank ( Table 1). Note that no TUB2 sequence was generated for the new species, A. bambusicola. Notwithstanding, this locus was included in our phylogenetic analyses to increase phylogenetic resolution. Sequences of individual loci were aligned using MAFFT v. 7 using the 'auto' option (https://mafft.cbrc.jp/alignment/ server/index.html) (Katoh et al. 2019) and, where necessary, improved in BioEdit v. 7.0.5.3 (Hall 1999. Multiple loci were combined by SequenceMatrix (Vaidya et al. 2011). The alignment was trimmed using trimAl v 1.2 with the 'gappyout' option (Capella-Gutiérrez et al. 2009). A phylogenetic tree was reconstructed from the concatenated ITS-LSU-TEF1-TUB2 dataset using Maximum Likelihood (ML), Maximum Parsimony (MP) and Bayesian Inference (BI) analyses. Phylogenetic analyses were performed using the CIPRES Science Gateway web portal (Miller et al. 2010). ML was done using the RAxML-HPC on XSEDE tool under the GTRGAMMA+I-Invar substitution model (Stamatakis et al. 2008). MP analysis was performed using the PAUP on XSEDE tool (Swofford 2002). A heuristic search with 1000 random taxa additions was used to infer MP trees. The value of MaxTrees was set to 5000, with branches of zero length collapsed and all multiple parsimonious trees saved.
Parsimony score values for tree length (TL), consistency index (CI), retention index (RI) and homoplasy index (HI) were calculated for trees generated under different optimum criteria. Robustness of branches was estimated by maximum parsimony bootstrap proportions, using 100 bootstrap replicates, with tree bisection-reconnection branch swapping and a re-arrangement limit of 1000.
BI analysis was performed using the MrBayes on XSEDE tool available on the CIPRES Science Gateway (Huelsenbeck and Ronquist 2001, Miller et al. 2010, Ronquist et al. 2012. The best-fit model for each locus was selected by MrModeltest version 2.3, under the Akaike Information Criterion. Four Markov Chain Monte Carlo (MCMC) chains were run, starting from a random tree topology. The operation was stopped automatically when the average standard deviation of split frequencies fell below 0.01. Markov chains were set to run 10,000,000 generations with sampling every 1000 generations. A burn-in set at 25% was discarded. The Maximum Clade Credibility tree was inferred with the highest product of separate clade posterior probabilities (

Phylogenetic analyses
The For the Bayesian posterior probabilities analysis, the best-fit models were selected as GTR+I+G for ITS and LSU and HKY+I+G for TEF1 and TUB2; 2,895,000 generations were run. A total of 2172 trees were maintained after discarding 25% as burnin. Bayesian PP were evaluated with a final average standard deviation of split frequencies of 0.009965.

Discussion
The family Apiosporaceae was introduced by Hyde et al. (1998) to accommodate Apiospora and Appendicospora, based on their unique morphology. Arthrinium is one of the asexual morphs of Apiospora, along with Cordella and Pteroconium (Hyde et al. 1998). Based on molecular evidence, Crous and Groenewald (2013) confirmed that the genus Arthrinium belongs to Apiosporaceae (Hyde et al. 2020b, Wijayawardene et al. 2020. Apiospora was shown to be synonymous with Arthrinium, which is the oldest name (Hawksworth et al. 2011, Crous andGroenewald 2013).
Arthrinium species have a highly-variable morphology Groenewald 2013, Dai et al. 2017). They produce hyphomycetous fungal structures in culture or coelomycetous fruiting bodies on their host, depending on the substrate and period of incubation Groenewald 2013, Dai et al. 2017). However, as more species of Arthrinium are discovered, identification, based on morphology alone, has become very difficult because some species exhibit similar micro-morphological characters ). Figs 2, 3 The best-scoring RAxML tree reconstructed from a concatenated ITS-LSU-TEF1-TUB2 dataset. The tree is rooted with Seiridium phylicae (strains CPC 19962 and CPC 19965   Arthrinium species have been reported from soil debris, plants, lichens, marine algae and hive-stored pollen (Senanayake et al. 2015, in the gut of insects (Crous et al. 2015), in nodules of human skin (Sharma et al. 2014) and especially associated with bamboo. To date, 24 Arthrinium species have been found in association with the bamboo subfamily Bambusoideae , Jiang et al. 2018, Jiang et al. 2020, https://nt.ars-grin.gov/fungaldatabases/). Arthrinium species have been reported from all continents, except Antarctica (Ellis 1963, Dyko and Sutton 1979, Calvo and Guarro 1980, Von Arx 1981, Crous and Groenewald 2013, Sharma et al. 2014, Jiang et al. 2020. To date, seven species of Arthrinium have been reported from Thailand. These are A. bambusicola (this study), A. chromolaenae (Mapook et al. 2020), A. longistromum , A. paraphaeospermum (Hyde et al. 2016), A. rasikravindrii ), A. subglobosum (Senanayake et al. 2015 and A. thailandicum . Contrasting morphological features amongst these species are presented in Suppl. material 1. Six of the seven Thai species are found in association with bamboo: Arthrinium bambusicola, A. longistromum, A. paraphaeospermum, A. rasikravindrii , A. subglobosum and A. thailandicum (Senanayake et al. 2015, Hyde et al. 2016. Only Arthrinium chromolaenae was reported from a non-bamboo host, Chromolaena odorata (Asterales, Asteraceae) (Mapook et al. 2020). Further studies on this genus in Thailand and other countries, as well as from different hosts, are likely to result in the discovery of more new species (Hyde et al. 2018, Hyde et al. 2020a).