Eight new Arthrinium species from China

Abstract The genus Arthrinium includes important plant pathogens, endophytes and saprobes with a wide host range and geographic distribution. In this paper, 74 Arthrinium strains isolated from various substrates such as bamboo leaves, tea plants, soil and air from karst caves in China were examined using a multi-locus phylogeny based on a combined dataset of ITS rDNA, TEF1 and TUB2, in conjunction with morphological characters, host association and ecological distribution. Eight new species were described based on their distinct phylogenetic relationships and morphological characters. Our results indicated a high species diversity of Arthrinium with wide host ranges, amongst which, Poaceae and Cyperaceae were the major host plant families of Arthrinium species.


Introduction
Arthrinium Kunze is an anamorph-typified genus, which has been traditionally linked to the teleomorph-typified genus Apiospora Sacc. (Ellis 1971, Seifert et al. 2011. It is strikingly different from other anamorphic genera for the presence of basauxic conidiophores (Hughes 1953, Minter 1985. The traditional generic circumscription of Arthrinium was primarily based on morphological characters (e.g. conidial shape, conidiophores, sterile cells and the presence of setae) but has been regarded as too narrow (Ellis 1971, Minter 1985, Crous et al. 2013. It is now recognised that, at the generic level, conidial shape and the presence of setae are not reliable characters to infer phylogenetic relationships (Crous et al. 2013). For example, Arthrinium was regarded as being different from Cordella Speg. (1886) by the absence of setae amongst the clusters of specialised hyphae and different from Pteroconium Sacc. (1892) by the absence of sporodochia and pseudoparenchyma (Minter 1985). However, both genera have been reduced to the generic synonyms of Arthrinium, based on molecular phylogenetic data (Crous et al. 2013).
Arthrinium species are geographically widely distributed in various hosts. Many species of Arthrinium are associated with plants as endophytes or saprobes, as well as plant pathogens on some important ornamentals, e.g. A. phaeospermum causing culm rot on Phyllostachys viridis (Li et al. 2016); A. arundinis causing brown culm streak of Phyllostachys praecox (Chen et al. 2014). Moreover, A. phaeospermum has been reported for causing cutaneous infections of humans (Rai 1989, Zhao et al. 1990, de Hoog et al. 2000, Crous et al. 2013. Many Arthrinium species are also known to produce bioactive compounds with pharmacological and medicinal applications, such as A. arundinis and A. saccharicola isolated from a brown alga Sargassum sp., with good antifungal activities against some plant pathogenic fungi (Hong et al. 2015). Arthrinium saccharicola, A. sacchari and A. phaeospermum isolated from Miscanthus sp. are known to produce industrially important enzymes (Shrestha et al. 2015).
In this paper, eight new Arthrinium species are described and characterised based on morphological characters and phylogeny inferred from the combined ITS rDNA, TEF1 and TUB2 sequences dataset. Comparisons were made with morphologically similar and phylogenetically related species. Fungus-host distribution of Arthrinium species are summarised based on data from literature and this study.

Materials and method
Isolates Diseased and healthy tissues of bamboo leaves and other plant hosts were collected from six provinces or municipalities in China (Chongqing, Guangxi, Guangdong, Guizhou, Jiangxi, Hunan). Tissue pieces (5 mm × 5 mm) were taken from the margin of leaf lesions and the surface sterilised with 75% ethanol for 1 min, 5% NaClO for 30 s, followed by rinsing in sterile distilled water for 1 min. The pieces were dried with sterilised paper towels and then placed on 1/4 PDA (potato dextrose agar) (Cai et al. 2009).
All cultures were preserved in the LC culture collection (personal culture collection of Lei Cai housed in the Institute of Microbiology, Chinese Academy of Sciences). Type specimens were deposited in Mycological Herbarium of the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS), with ex-type living cultures deposited in China General Microbiological Culture Collection Center (CG-MCC). Taxonomic information of the new taxa was deposited in MycoBank (www. MycoBank.org; Crous et al. 2004).

Morphology
Cultures were incubated on PDA for 7 d at 25 °C to measure the growth rates and on 2% malt agar with bamboo leaves to enhance sporulation. Morphological descriptions were based on cultures sporulating on MEA (malt extract agar) medium at room temperature (ca. 25 °C). Shape and size of microscopic structures were observed using a light microscope and colonies were assessed according to the colour charts of Rayner (1970). At least 50 conidiogenous cells and conidia were measured to calculate the mean size.

DNA extraction, PCR amplification and sequencing
Fresh fungal mycelia were taken from 7-d-old cultures growing on PDA and ground with the organisation disruptor FastPrep-48. Genomic DNA was extracted following the modified CTAB protocol as described in Guo et al. (2000).

Phylogenetic analysis
Sequences generated from the forward and reverse primers were used to obtain consensus sequences using MEGA v. 6.0 (Tamura et al. 2013). The concatenated tree was inferred based ITS, TUB2 and TEF1 sequences ( Figure 1) using Bayesian and Maximum-likelihood analyses. Sequences were aligned using an online version of MAFFT v. 7 (available at http://mafft.cbrc.jp/alignment/server/). Ambiguous regions were excluded from the analyses and gaps were treated as missing data. Maximum-likelihood (ML) analysis was performed in RAxML v. 7.2.6 (Stamatakis and Alachiotis 2010), employing GTR models of evolution settings of the programme and bootstrap support obtained by running 1000 pseudo replicates. Maximum Likelihood bootstrap values (ML) equal to or greater than 70% are given above each node.
Bayesian analysis was conducted using MrBayes v. 3.2.1 (Ronquist et al. 2012) and the best nucleotide substitution model for each locus was calculated with jModelTest v. 2.1.4 (Posada 2008). Posterior probabilities (PP) (Zhaxybayeva and Gogarten 2002) were de-Figure 1. Phylogenetic tree based on the combined ITS, TEF1 and TUB2 sequences alignment generated from a Maximum likelihood phylogenetic analysis. Bootstrap support values (>70%) and posterior probabilities (>0.9) are given at the nodes (ML/PP). The tree is rooted with Nigrospora gorlenkoana CBS 480.73. The novel species were highlighted (* indicates the ex-type cultures).
termined by Markov Chain Monte Carlo sampling (MCMC) under the estimated model of evolution. Four simultaneous Markov chains were run for 10 million generations and trees were sampled every 1000 generations. The run was stopped automatically when the average standard deviation of split frequencies fell below 0.01. The first 25% trees, which represented the burn-in phase of the analyses, were discarded and the remaining trees were used for calculating PP in the majority rule consensus tree. Sequences generated in this study were deposited in GenBank (Table 1) and the final matrices used for the phylogenetic analyses in TreeBASE (www.treebase.org; accession number: 21341).

Fungus-host distribution of Arthrinium species
To determine the distribution of Arthrinium species on host/substrate, the number of species occurred on each host (based on family level) was counted based on data from this study, relevant literature and the USDA fungal database (https://nt.ars-grin.gov/ fungaldatabases/). The proportion account for the known 66 species in Arthrinium (Index Fungorum) was illustrated in a histogram. Four species with an unknown host range were not included in this analysis.

Phylogeny
The combined ITS, TUB2 and TEF1 dataset contained 75 strains, with Nigrospora gorlenkoana CBS 480.73 as the out group. For the Bayesian analyses, the best-fit models TrN+I+G, GTR+I+G, HKY+I+G were selected for ITS, TUB2 and TEF1 loci, respectively. The ML analysis showed the same tree topology as that obtained in the Bayesian analysis. All the Arthrinium strains in this study separated into 13 clades, representing five known (A. arundinis, A. hydei, A. rasikravindrii, A. thailandicum, A. xenocordella) and eight new species (Figure 1). The eight new species clustered in distinct clades with high bootstrap supports ( Figure 1). Phylogenetic analyses based on an individual locus were also conducted (not shown) and the generated trees are similar to the one generated from the combined multi-locus dataset ( Figure 1).
Culture characteristics. On PDA, colonies flat, spreading, margin circular, with abundant aerial mycelia, surface and reverse white to grey. On MEA, colonies flat, spreading, surface and reverse brown to black.
Culture characteristics. On PDA, colonies flat, margin circular, initially white, becoming greyish on surface, reaching 9 cm in 7 days at 25 °C. On MEA, with sparse aerial mycelia, surface dirty white, reverse pale luteous.
Culture characteristics. On PDA, colonies flat, woolly, margin circular, with moderate aerial mycelia, surface initially white, becoming greyish and reverse with black patches, reaching 9 cm in 9 days at 25 °C. On MEA, surface dirty white with patches of olivaceous-grey and reverse greyish.
Culture characteristics. On PDA, colonies flat, woolly, margin circular, with sparse aerial mycelia, initially white, becoming greyish due to sporulation, reaching 9 cm in 10 days at 25 °C, on MEA, sienna with patches of luteous, reverse luteous to sienna.

Arthrinium obovatum
Culture characteristics. On PDA, colonies flat, spreading, margin circular, initially white, becoming olivaceous-grey on surface, reverse smoke-grey with patches of olivaceous grey, reaching 9 cm in 7 days at 25 °C. On MEA, surface olivaceous grey in the central and luteous around, reverse with patches of olivaceous grey.
Culture characteristics. On PDA, colonies flat, spreading, margin circular, with moderate aerial mycelia, initially white, becoming grey on surface, reverse smoke-grey without patches, reaching 9 cm in 8 days at 25 °C. On MEA, surface pale luteous to grey with abundant mycelia, reverse greyish without patches.
Culture characteristics. On PDA, colonies flat, spreading, margin circular, with moderate aerial mycelia, initially white, becoming light pink on surface, reverse peachpuff without patches, reaching 10 cm in 8 days at 25 °C. On MEA, surface blackishgreen with abundant mycelia, reverse with patches of greyish.
Notes. Three strains representing A. subroseum clustered in a well-supported clade, closely related to A. garethjonesii (94% sequence similarity in ITS) and A. bambusae (98% sequence similarity in ITS; 92% in TUB2; 96% in TEF1). However, A. subro-seum differs from A. bambusae in the morphology of conidiophores (erect or ascending, clustered in groups in A. subroseum vs. reduced to conidiogenous cells in A. bambusae). Arthrinium subroseum is not morphologically comparable to A. garethjonesii, whose asexual morph is undetermined (Dai et al. 2016b).

Discussion
Arthrinium, Cordella and Pteroconium share similar morphological characters, e.g. basauxic conidiophores with terminal and intercalary polyblastic conidiogenous cells and brown, unicellular conidia with a pallid germ slit (Ellis 1971, Hyde et al. 1998). Crous et al. (2013) reduced both Cordella and Pteroconium as generic synonyms of Arthrinium based on molecular phylogenetic data and regarded traditionally applied morphological characters in distinguishing these genera as phylogenetically insignificant. This study added eight novel species and our data are in good accordance with that of Crous et al. (2013). For example, A. pseudoparenchymaticum is sporodochial and pseudoparenchymatous, which would be classified as Pteroconium in the traditional taxonomy. However, the multi-locus (ITS, TEF1 & TUB2) tree ( Figure. 1) shows that A. pseudoparenchymaticum is phylogenetically distant from A. pterospermum (syn. P. pterospermum, the type of "Pteroconium").
Currently there are 70 recognised species in Arthrinium (Index Fungorum), occurring on a wide variety of both living and decaying plant materials. It is noteworthy that Arthrinium species showed distinct preference for growing on two graminaceous families, Poaceae and Cyperaceae, amongst which, Bambusa (Poaceae) and Carex (Cyperaceae) are two of the most common host genera for Arthrinium species. For example, seven species have been recorded from Carex spp., i.e. A. austriacum Petr. (1959), A. caricicola Kunze (1817), A. globosum Koskela (1983), A. kamtschaticum Tranzschel & Woron (1914), A. morthieri Fuckel (1870), A. muelleri Ellis (1976) andA. naviculare Rostr. (1886). Bamboo has been widely known as a favourable host for Arthrinium, e.g. A. hyphopodii, A. longistromum, A. subglobosum, A. thailandicum andA. yunnanum (Senanayake et al. 2015, Dai et al. 2016). In this study, three new species (A. bambusae, A. subroseum and A. pseudoparenchymaticum) were also isolated from bamboo. In addition, three species (A. arundinis, A. guizhouense, and A. rasikravindrii) were isolated from air and soil from karst caves, where have been shown to encompass a high fungal diversity (Jiang et al. 2017, Zhang et al. 2017. In addition to the Arthrinium species from China, we also tried to resolve the phylogenetic status of Arthrinium mytilomorphum Bhat & W.B. Kendr. (Bhat 1993) in the current study. DNA extraction from the type specimen of A. mytilomorphum (DAOM 214595) was prohibited but DAOM provided a DNA sample. Unfortunately, we only managed to obtain an ITS sequence from this DNA sample, while the amplifications of all other protein coding genes were unsuccessful. The ITS phylogenetic tree (not shown here) shows that A. mytilomorphum is closely related to A. subroseum (99 % sequence similarity in ITS), while the morphology of these two species are very different from each other. Conidia of A. mytilomorphum are dark brown, fusiform or navicular, measuring 20-30 × 6-8.5 µm, slightly bowed down and asymmetric (Figure 11), while those of A. subroseum are pale brown to dark brown, globose or subglobose, measuring 12-17.5 × 9-16 µm. Teleomorph-typified genus Apiospora was treated as a synonym of anamorphtypified genus Arthrinium on the basis that Arthrinium is older and more commonly used in literature (Crous et al. 2013). However, only three of the 58 recorded Apiospora species have been properly linked to their known Arthrinium counterparts, i.e. Arthrinium hysterinum (syn. Ap. bambusae) (Sivanesan 1983, Kirk 1986); Arthrinium arundinis (syn. Ap. montagnei) (Hyde 1998); Arthrinium sinense (syn. Ap. sinensis) (Réblová et al. 2016). In addition, molecular data of only four Apiospora species (Ap. bambusae, Ap. montagnei, Ap. setosa and Ap. sinensis) are available, in which only A. bambusae and A. sinensis have type-derived sequences. A comprehensive taxonomic revision of this taxonomic group awaits fresh collection and epitypification of many Apiospora species and, based on which, phylogenetic links with Arthrinium species could be established.