Neostagonosporellasichuanensis gen. et sp. nov. (Phaeosphaeriaceae, Pleosporales) on Phyllostachysheteroclada (Poaceae) from Sichuan Province, China

Abstract Neostagonosporellasichuanensis sp. nov. was found on Phyllostachysheteroclada collected from Sichuan Province in China and is introduced in a new genus Neostagonosporella gen. nov. in this paper. Evidence for the placement of the new taxon in the family Phaeosphaeriaceae is supported by morphology and phylogenetic analysis of a combined LSU, SSU, ITS and TEF 1-α DNA sequence dataset. Maximum-likelihood, maximum-parsimony and Bayesian inference phylogenetic analyses support Neostagonosporella as a distinct genus within this family. The new genus is compared with related genera of Phaeosphaeriaceae and full descriptions and illustrations are provided. Neostagonosporella is characterised by its unique suite of characters, such as multiloculate ascostromata and cylindrical to fusiform, transversely multiseptate, straight or curved ascospores, which are widest at the central cells. Conidiostromata are multiloculate, fusiform to long fusiform or rhomboid, with two types conidia; macroconidia vermiform or subcylindrical to cylindrical, transversely multiseptate, sometimes curved, almost equidistant between septa and microconidia oval, ellipsoidal or long ellipsoidal, aseptate, rounded at both ends. An updated phylogeny of the Phaeosphaeriaceae based on multigene analysis is provided.


Introduction
The family Phaeosphaeriaceae is a large and important family of Pleosporales, initially introduced by Barr (1979) with Phaeosphaeria oryzae I. Miyake as the type species (Miyake 1909). The taxonomy of members within this family has often been confused with those of the Leptosphaeriaceae (Müller 1950, Holm et al. 1957, Munk 1957 and it is sometimes difficult to distinguish species. Criteria which have previously been used to differentiate species have been based mostly on the morphology of the peridial wall, asexual characteristics and host association (Eriksson 1967, 1981, Lucas and Webster 1967, Leuchtmann 1984, Shoemaker 1984, Barr 1987, Shoemaker and Babcock 1989, Shearer et al. 1990, Khashnobish and Shearer 1996, Câmara et al. 2002 and taxonomic schemes followed are those of Kirk et al. (2008), Zhang et al. (2009), Hyde et al. (2013), Phookamsak et al. (2014a) and Abd-Elsalam et al. (2016). However, this delimitation of taxa in Phaeosphaeriaceae and Leptosphaeriaceae, based solely on the above-mentioned features, is not feasible. Recent studies showed that it is very difficult to discriminate them only by such characters, because numerous new members have been introduced to these two families and these species are not significantly different in these features, but they can be differentiated by phylogenetic analysis (Zhang et al. 2012, Ahmed et al. 2014, Ariyawansa et al. 2015a, Bakhshi et al. 2018. Hence there is a need to use the multigene sequence data analyses to infer relationships. Barr (1979) originally introduced 15 genera in this family and subsequent researchers have revised this number (Barr 1992, Eriksson and Hawksworth 1993, Kirk et al. 2001, Lumbsch and Huhndorf 2007. The taxonomic placement of genera within this family has been changed in recent years based on phylogenetic analyses (Zhang et al. 2012, Wijayawardene et al. 2014, Phookamsak et al. 2014a, Wanasinghe et al. 2018. Taxonomic revision of the genera in Phaeosphaeriaceae resulted in 28 genera based on morphology and phylogenetic evidence (Phookamsak et al. 2014a). Since 2014, many new genera have been introduced based on molecular data (Ariyawansa et al. 2015b, Ertz et al. 2015, Crous et al. 2015a, 2017a, Rossman et al. 2015, Abd-Elsalam et al. 2016, Hernández-Restrepo et al. 2016, Ahmed et al. 2017, Bakhshi et al. 2018, Wanasinghe et al. 2018. The placement of some older genera has been reconfirmed with DNA sequence . However, there are still a few genera lacking molecular data, such as Bricookea, Dothideopsella, Eudarluca, Phaeostagonospora and Tiarospora. At present, this family includes more than 800 species in 61 genera (25 genera are known only from asexual morphs) (Index Fungorum 2018, Wijayawardene et al. 2017Wijayawardene et al. , 2018. Many genera were introduced to accommodate a single or a few species in Phaeosphaeriaceae. Only 14 genera in the Phaeosphaeriaceae contained 10-50 species, while Ophiobolus and Phaeosphaeria comprised more than 150 species. However, most species in Ophiobolus and Phaeosphaeria lack molecular data to confirm their phylogenetic affinities. We are studying fungi on bamboo which is the main food for panda in Sichuan Province of China (Tang et al. 2007. The purpose of this paper is to introduce a new genus with one species in Phaeosphaeriaceae recovered from Phyllostachys heteroclada Oliv. Combined multigene (LSU, SSU, ITS and TEF 1-α) analyses confirm its phylogenetic position in Phaeosphaeriaceae. A comprehensive comparison with similar genera and detailed descriptions and illustrations are provided.

Sampling and morphological study
The specimens were collected from Ya'an City of Sichuan Province in China, on living to near dead stems and branches of Phyllostachys heteroclada. The samples were kept in Ziplock plastic bags and brought to the laboratory. Fresh materials were examined by using stereo and compound microscopes. Vertical free-hand sections were made by using a razor blade and placed on a droplet of sterilised water on a glass slide (Gupta and Tuohy 2013). Lactate cotton blue reagent was used to observe the number of septa. Micromorphological characters were examined by using a Nikon ECLIPSE Ni compound microscope fitted to a Cannon 600D digital camera. Fruiting tissues were observed by stereomicroscopy using NVT-GG (Shanghai Advanced Photoelectric Technology Co. Ltd, China) and photographed by VS-800C (Shenzhen Weishen Times Technology Co. Ltd, China). Measurements were taken using Tarosoft® Image Frame Work v.0.9.7.

Isolation
Single ascospore and conidium isolation was carried out following the method described by Dai et al. (2017). Germinated ascospores and conidia were separately transferred to Potato Dextrose Agar media plates (PDA) and incubated at 25°C and the colonies were observed after 10 days and as outlined by Vijaykrishna et al. (2004) and Liu et al. (2010). Specimens are deposited in Mae Fah Luang University Herbarium (MFLU), Chiang Rai, Thailand and Sichuan Agricultural University Herbarium (SICAU), Chengdu, China. Living cultures are deposited at the Culture Collection at Mae Fah Luang University (MFLUCC) and the Culture Collection at Sichuan Agricultural University (SICAUCC). Facesoffungi and Index Fungorum numbers were registered as in Jayasiri et al. (2015) and Index Fungorum (2018), respectively. New species are established following the recommendations of Jeewon and Hyde (2016).

DNA extraction, PCR amplification and sequencing
Fungal isolates were grown on PDA for seven days at 25°C and genomic DNA was extracted from fresh mycelia, following the protocols of Plant Genomic DNA Kit (Tiangen, China). If cultures were unavailable, fungal DNA was directly extracted from fruiting tissues according to Yang et al. (2017), Wanasinghe et al. (2018) and . The primers, LR0R and LR5 (Vilgalys and Hester 1990), NS1 and NS4, ITS5 and ITS4 (White et al. 1990) and EF1-983F and EF1-2218R (Rehner 2001) were used for the amplification of the 28S large subunit rDNA (LSU), 18S small subunit rDNA (SSU), internal transcribed spacers (5.8S, ITS) and translation elongation factor 1-α gene region (TEF 1-α), respectively. The amplification reactions were performed as stated by Phukhamsakda et al. (2015). Amplified PCR fragments were purified and sequenced at TsingKe Biological Technology Co., Ltd. (Chengdu, China). Newly generated sequences of LSU, SSU, ITS and TEF 1-α regions are deposited in GenBank.
Maximum likelihood analysis was generated by using the CIPRES Science Gateway web server (Miller et al. 2010) and chosen RAxML-HPC BlackBox (8.2.10) (Stamatakis 2014). Maximum parsimony analysis was performed by PAUP v. 4.0b10 (Swofford 2002) with the heuristic search option with 1,000 random sequence additions and treebisection reconnection (TBR) as branch-swapping algorithm. All characters were unordered and of equal weight and gaps were regarded as missing data. Maxtrees were set up to 1,000, a zero of maximum branches length was collapsed and all multiple parsimonious trees were saved.   [HI] were determined under different optimality criteria. The robustness was assessed using bootstrap analysis with 1,000 replications (Hillis and Bull 1993). The Kishino-Hasegawa tests were made in order to determine whether trees were significantly different (Kishino and Hasegawa 1989).
Bayesian inference analysis was conducted with MrBayes v. 3.2.2 (Ronquist et al. 2012) and a Bayesian posterior probability (BYPP) was determined by Markov Chain Monte Carlo sampling (MCMC). The Bayesian parameters were set up to "Lset ap-plyto= (all) nst=6 rates=invgamma; prset applyto= (all) statefreqpr=dirichlet (1,1,1,1)". Six simultaneous Markov chains were set up to 10,000,000 generations and trees were sampled every 100 th generation. The programme was automatically terminated when the average standard deviation of split frequencies reached below 0.01 . The distribution of log-likelihood scores were examined to determine the stationary phase for each search and to decide if extra runs were required to achieve convergence, using Tracer v.1.6 program (Rambaut et al. 2013). The first 10% of generated trees representing the burn-in phase were discarded and the remaining trees were used to calculate posterior probabilities of the majority rule consensus tree.
Notes. Ex-type strains are given in bold and the new species in this study is in red. "-" means that the sequence is missing or unavailable. Abbreviations

Phylogenetic analyses
In this phylogenetic analysis, we include all representative sequences of genera in Phaeosphaeriaceae and other representative genera and species in Pleosporineae and Massarineae. The final concatenated dataset containing 138 ingroup taxa within the suborder Pleosporineae, included 56 currently existing genera in Phaeosphaeriaceae, with 3559 characters including gaps (917 characters for LSU, 1046 for SSU, 681 for ITS and 915 for TEF 1-α). Single gene datasets of LSU, SSU, ITS and TEF 1-α were  initially analysed and checked for topological congruence but these were not significantly different (data not shown). Support values of MP, ML and BI analyses (equal to or higher than 70% for MPBP and MLBP and 0.95 for BYPP) are shown in Fig.  1 which is the best scoring tree generated from ML. The phylogenetic trees generated from ML analyses were similar to previous phylogenies including Phaeosphaeriaceae (Phookamsak et al. 2014a, Ahmed et al. 2017, Ariyawansa et al. 2018, Bakhshi et al. 2018, Wanasinghe et al. 2018. The best scoring RAxML tree with the final optimisation had a likelihood value of -32702.569414. The matrix had 1387 distinct alignment patterns and 32.39% in this alignment is the gaps and completely undetermined characters. Estimated base frequencies were as follows: A=0.244424, C=0.233850, G=0.265929, T=0.255797, with substitution rates AC=1.171601, AG=2.805496, AT=2.145028, CG=0.771605, CT=6.035018 and GT=1.000000. The gamma distribution shape parameter α=0.167161 and the Tree-Length=5.334112. The maximum parsimony dataset con- sisted of 3559 characters, of which 2580 characters were constant, 217 were parsimony-uninformative and 762 were parsimony-informative. All characters were of type 'unord' with equal weight. The parsimony analysis resulted in a thousand equally most parsimonious trees with a length of 5829 steps (CI = 0.270, RI = 0.654, RC = 0.177, HI = 0.730). Bayesian posterior probabilities were determined by MCMC and the final average standard deviation of split frequencies was 0.009939.
Neostagonosporella sichuanensis clusters in the family Phaeosphaeriaceae with strong support (100% MLBP/100% MPBP/1.00 BYPP) and nucleotide sequences from all strains are the same and it confirms that our three collections are the same species. The multigene analyses show that N. sichuanensis is phylogenetically close to the genus Setophoma and Edenia and separated from the remaining genera of the family in a distinct clade with moderate bootstrap support. Etymology. Name reflects the morphological similarity to the genus Stagonospora. Description. Parasitic on living to nearly dead stems and branches of bamboo. Sexual morph: Ascostromata coriaceous, visible as raised to superficial on host, gregarious, multi-loculate, ellipsoidal, globose to subglobose or irregular in shape, dark brown to black, glabrous. Locules globose to subglobose, with a centrally located ostiole, lacking periphyses. Peridium multi-layered, of brown to dark brown, pseudoparenchymatous cells of textura angularis. Hamathecium comprising trabeculate, anastomosed pseudoparaphyses. Asci 8-spored, bitunicate, fissitunicate, cylindrical to cylindric-clavate, short pedicellate, apically rounded with an ocular chamber. Ascospores overlapping biseriate, hyaline, cylindrical to fusiform, septate, smooth-walled, surrounded by a distinct mucilaginous sheath. Asexual morph: Coelomycetous. Conidiostromata pycindial, coriaceous, superficial, dark brown to black, fusiform to long fusiform or rhomboid, multi-loculate, solitary, glabrous. Pycnidia globose to subglobose, ostiolate. Pycnidial wall comprising multi-layered, of dark brown to black, pseudoparenchymatous cells of textura angularis. Conidiophores reduced to conidiogenous cells. Conidiogenous cells ampulliform to subcylindrical, smooth, hyaline, enteroblastic, phialidic, arising from inner layer of pycnidial wall. Macroconidia hyaline, subcylindrical to cylindrical, septate, nearly equidistant between septa, smooth-walled, sometimes surrounded by a mucilaginous sheath when immature. Microconidia hyaline, varied in shape, aseptate, smooth-walled, with small guttulate.

Discussion
Neostagonosporella has a unique suite of characters that differentiate it from other genera in Phaeosphaeriaceae, such as multi-loculate ascostromata and trabeculate pseudoparaphyses. Trabeculate pseudoparaphyses have been shown to be uninformative at the higher taxonomic levels (Liew et al. 2000), but appear to be informative at the genus level. Neostagonosporella is the only genus of Phaeosphaeriaceae with this type of pseudoparaphyses. Phaeosphaeriaceous taxa have diverse morphological characteristics and the familial placement of some genera could not be resolved based on a concatenated phylogeny of three to four loci, because some genera contain only 1-2 described species (Crous et al. 2015a, 2017a, Abd-Elsalam et al. 2016, Hernández-Restrepo et al. 2016, Ahmed et al. 2017, Bakhshi et al. 2018, Wanasinghe et al. 2018. Species of Phaeosphaeriaceae have been found on various hosts and substrates, including plants, lichens, mushrooms, algae, human, soil and air (Saccardo 1883, Berlese and Voglino 1886, Phookamsak et al. 2014a, Ahmed et al. 2016, Joshi et al. 2018). However, most Phaeosphaeriaceous genera occur on plants of more than 65 host families, the majority of them being monocotyledons and herbaceous plants, such as Arecaceae, Asparagaceae, Compositae, Juncaceae, Leguminosae, Poaceae, Ranunculaceae, Restionaceae and Rosaceae etc. (Taylor and Hyde 2003, Quaedvlieg et al. 2013, Crous et al. 2015b, Tibpromma et al. 2016a, Wanasinghe et al. 2018. Our new genus exists on Poaceae and at least 30 genera are reported within this family. Currently, 11 genera are observed only on Poaceae: Amarenomyces, Bricookea, Camarosporioides, Dactylidina, Embarria, Melnikia, Neosphaerellopsis, Phaeopoacea, Sulcispora, Vagicola and Yunnanensis, all of them being recently established except for Amarenomyces, Bricookea and Sulcispora (Eriksson 1981, Barr 1982, Shoemaker and Babcock 1989, Trakunyingcharoen et al. 2014, Ariyawansa et al. 2015b, Wanasinghe et al. 2018. Amongst them, all hosts are short herbaceous plants and there are no bamboo plants recorded so far, with the exception of a few species of Ophiobolus and Phaeosphaeria in the old literature (Penzig andSaccardo 1897, Miyake andHara 1910). A large number of bamboo forests (more than 130 species) are distributed throughout Sichuan (Yi 1997) and, most likely, many Phaeosphaeriaceae species are waiting for exploration and discovery.