Bambusicolous Arthrinium Species in Guangdong Province, China

A survey of bambusicolous fungi in Bijiashan Mountain Park, Shenzhen, Guangdong Province, China, revealed several Arthrinium-like taxa from dead sheaths, twigs, and clumps of Bambusa species. Phylogenetic relationships were investigated based on morphology and combined analyses of the internal transcribed spacer region (ITS), large subunit nuclear ribosomal DNA (LSU), beta tubulin (β-tubulin), and translation elongation factor 1-alpha (tef 1-α) gene sequences. Based on morphological characteristics and phylogenetic data, Arthrinium acutiapicum sp. nov. and Arthrinium pseudorasikravindrae sp. nov. are introduced herein with descriptions and illustrations. Additionally, two new locality records of Arthrinium bambusae and Arthrinium guizhouense are described and illustrated.


INTRODUCTION
Arthrinium Kunze is accommodated in Apiosporaceae, Xylariales, which is morphologically different from other xylariaceous genera by the presence of basauxic conidiophores and dark, aseptate, globose to lenticular conidia with a hyaline rim or germ slit (Minter, 1985;Petrini and Müller, 1986;Singh et al., 2012;Jiang et al., 2018;Pintos et al., 2019). Basauxic conidiophores simply mean elongation of conidiogenous cells from the basal growing point after formation of a single, terminal blastic conidium at its apex (Cole, 1986).
Several studies revealed bambusicolous Arthrinium species from Poaceae and Cyperaceae host plants in China (Dai et al., 2016, 2017Jiang et al., 2018Jiang et al., , 2020Wang et al., 2018), and several Arthrinium-like taxa on dead leaves, clumps, and twigs of bamboo were collected from Shenzhen (China) during this study. The aims of this study are identifying these Arthrinium-like taxa based on morphology and phylogeny and describe and illustrate them. Phylogenetic relationships were investigated based on DNA sequence data of the internal transcribed spacer region (ITS), large subunit nuclear ribosomal DNA (LSU), beta tubulin (β-tubulin), and translation elongation factor 1-alpha (tef 1-α), and two new Arthrinium species from bamboo are introduced as Arthrinium pseudorasikravindrae and A. acutiapicum and two locality records, Arthrinium bambusae and Arthrinium guizhouense, are described and illustrated.

Sample Collection and Fungal Isolation
Fresh specimens of Arthrinium-like taxa were collected from Bijiashan Mountain Park, Shenzhen, Guangdong Province, China, in September-October 2018, and the collection site has a tropical climate with abundant sunshine and rainfall all year round. The yearly average temperature is 22 • C and vegetative type is tropical evergreen monsoon forests (Li et al., 2015). Specimens were brought to the laboratory in paper bags and they were examined under a stereomicroscope (Carl Zeiss Discovery V8), and blackish conidial mass and fruit bodies were observed. The fruit bodies were studied and photographed using a stereomicroscope fitted with a camera (ZEISS Axiocam 208). The micromorphological characters were studied and photographed using a compound microscope (Nikon Eclipse 80i) fitted with a digital camera (Canon 450D). All microscopic measurements such as the length of the conidiophores, conidiogenous cells, and conidia were made with Tarosoft image framework (v. 0.9.0.7).
Single conidial isolation was carried out following the method described by Senanayake et al. (2020a). Germinated conidia were aseptically transferred into fresh potato dextrose agar (PDA) plates, incubated at 20 • C to obtain pure cultures, and later transferred to PDA slants and stored at 4 • C for further study. Colony characteristics were recorded from cultures grown on PDA. Additionally, pure cultures were inoculated in 2% PDA together with sterilized bamboo leaves and incubated at 25 • C for sporulation.

DNA Extraction, PCR Amplification, and Sequencing
Fresh fungal mycelium grown on PDA for 2 weeks at 20 • C in the dark was used for DNA extraction using fungal genomic DNA extraction kit (Biospin DNA Extraction Kit, Bioer Technology, Co. Ltd., Hangzhou, China) following the manufacturer's protocols. Polymerase chain reactions (PCR) and sequencing were carried out for the following loci: the complete ITS region with the primer pair of ITS1/ITS4 (White et al., 1990); the LSU ribosomal DNA, amplified and sequenced as a single fragment with primers LR0R/LR5 (Vilgalys and Hester, 1990); the tef 1-α gene with primers EF1-728F/EF2 (Carbone and Kohn, 1999;Rehner, 2001); and the β-tubulin gene with primers bt2a and bt2b (Glass and Donaldson, 1995).
The PCR amplification reactions were carried out with the following protocol. The total volume of the PCR reaction was 25 µl reaction volume containing 1 µl of DNA template, 1 µl of each forward and reverse primer, 12.5 µl of 2 × PCR Master Mix, and 9.5 µl of double-distilled sterilized water (ddH 2 O). The reaction was conducted by running for 35 cycles following the condition of Senanayake et al. (2020b). The PCR products were observed on 1% agarose electrophoresis gel stained with ethidium bromide. Purification and sequencing of PCR products were carried out at Sunbiotech Company, Beijing, China. Sequence quality was checked and sequences were condensed with DNASTAR Lasergene v.7.1. Sequences derived in this study were deposited in GenBank and accession numbers were obtained (Table 1).

Sequence Alignments and Phylogenetic Analyses
BLASTn searches were made using the newly generated sequences to assist in taxon sampling for phylogenetic analyses. Jiang et al. (2018Jiang et al. ( , 2020, Wang et al. (2018), and Pintos et al. (2019) were followed to obtain sequences from GenBank that are listed in Table 1. The concatenated ITS, LSU, β-tubulin, and tef 1-α sequence dataset comprised 101 strains of Arthrinium, while the outgroup taxon was Pestalotiopsis chamaeropis (CBS 237.38). DNA sequences of the ITS, LSU, β-tubulin, and tef 1-α were aligned using the online version of MAFFT v. 7.036 2 (Katoh et al., 2020) with default settings and manually adjusted using BioEdit 7.1.3 (Hall, 1999) to allow maximum alignment and minimum gaps. Further, single gene alignments were combined to obtain the final multiloci alignment that was containing 2,817 nucleotide characters, viz. 681 of ITS, 875 of LSU, 434 of β-tubulin, and 827 of tef 1-α. Both single and concatenated alignments were used for the analyses.   Maximum likelihood analyses were performed by RAxML (Stamatakis and Alachiotis, 2010) implemented in raxmlGUIv.1.5 (Silvestro and Michalak, 2012) using the ML + rapid bootstrap setting and the GTR + I + G model of nucleotide substitution with 1,000 replicates. The matrix was partitioned for the different gene regions included in the combined multilocus analyses.
For the Bayesian inference (BI) analyses, the optimal substitution model for the combined dataset was determined to be GTR + I + G using the MrModeltest software v. 2.2 (Nylander, 2004). The BI analyses was computed with MrBayes v. 3.2.6 (Ronquist et al., 2012) with four simultaneous Markov chain Monte Carlo chains from random trees over 10 M generations (trees were sampled every 500th generation).
The distribution of log-likelihood scores was observed to check whether sampling is in stationary phase or not, and Tracer v1.5 was used to check if further runs were required to reach convergence or not (Rambaut and Drummond, 2007). The Bayesian analyses lasted until the average standard deviation of split frequencies has a value less than 0.01, and the consensus tree and posterior probabilities were calculated after discarding the first 20% of the sampled trees as burn-in. The phylogram was visualized in FigTree v. 1.4 (Rambaut, 2009). All the phylogenetic trees derived from this study were deposited in TreeBase 3 under accession number S27147.

Phylogenetic Inferences
All individual trees generated under different criteria and from single gene datasets were essentially similar in topology and not significantly different from the tree generated from the concatenated dataset (not discussed herein). Additionally, this tree topology is similar to previous studies on Arthrinium (Dai et al., 2016;Jiang et al., 2018Jiang et al., , 2020Wang et al., 2018;Pintos et al., 2019).
Maximum likelihood analysis of Arthrinium species in this study with 1,000 bootstrap replicates yielded the best ML tree (Figure 1) with the likelihood value of -29,933.362493 and the following model parameters: estimated base frequencies-A = 0.239654, C = 0.250345, G = 0.255054, and T = 0.254948; substitution rates-AC = 1.275584, AG = 2.530572, AT = 1.397969, CG = 1.184045, CT = 4.063803, and GT = 1.0; proportion of invariable sites-I = 0.203121; gamma distribution shape parameter-α = 0.54383. The alignment contained a total of 1,756 distinct alignment patterns and 28.72% of undetermined characters. After discarding the first 20% of generations, 36,000 trees remained from which 50% consensus trees and posterior probabilities (PP) were calculated (Figure 1). Maximum likelihood bootstrap values ≥ 60% and BI ≥ 0.95 are given at each node. Tree topologies of the ML and Bayesian analyses were similar to each other and there are no significant differences.
There are 101 Arthrinium strains in this study together with a new isolate that is introduced here. All the ex-type strains of Arthrinium species were included if available, and other authentic strains were selected when sequences from ex-type strains are unavailable. Our new isolate KUMCC 20-0206 clustered with the type strain of A. guizhouense (CGMCC 3.18334) and another representative strain (LC5318) with 87% ML and 0.95 PP support. This clade (clade A) has a close phylogenetic affinity to Arthrinium longistromum, A. piptatheri, and A. sacchari with 95% ML and 0.95 PP support. Two strains of A. bambusae (CGMCC 3.18335 and LC7107) and the new isolate KUMCC 20-0207 were grouped in a separate clade with 96% ML and 1.00 PP support. This clade (subclade B1) shares a monophyletic relationship to Arthrinium garethgonesii, A. mytilomotphum, A. neogarethjonesii, A. setostromun, and A. subroseum with strong bootstrap supports (100% ML, 1.00 PP, clade B, Figure 1). Two new isolates, KUMCC 20-0208 and KUMCC 20-0211, were monophyletic in subclade C1 (Figure 1) with 90% ML and 0.96 PP support. Subclade C2 is also monophyletic with two novel strains, viz. KUMCC 20-0209 and KUMCC 20-0210, which are sisters to subclade C1 with 90% ML and 0.96 PP support (clade C, Figure 1). With these four new strains, clade C shares a close phylogenetic affiliation to A. paraphaeospermum and A. rasikravindrae.

Culture Characteristics
Colonies grew on PDA at 20 • C in the dark attenuated 2 cm diam., within 7 days, flat, circular, entire margin, wooly, with abundant aerial mycelia, white in surface view and off-white to yellow in reverse. Sporulation occurred after 10 days on PDA incubated at 20 • C in the dark without any host substrate. Conidia seem black mass and well spread on culture.

Notes
Arthrinium acutiapicum forms a distinct subclade (subclade C2, Figure 1) with strong bootstrap support values (ML/PP = 90/0.96) in our phylogenetic analysis, which is a sister to the newly introduced species A. pseudorasikravindrae.
Additionally, A. acutiapicum shows close phylogenetic affinities to A. paraphaeospermum, A. pseudorasikravindrae, and A. rasikravindrae in clade C (Figure 1) and A. chinense. Blast results of ITS, LSU, β-tubulin, and tef 1-α sequences of A. acutiapicum show high similarity to A. hydei, A. paraphaeospermum, and A. rasikravindrae. Morphologically, A. acutiapicum is distinct from A. pseudorasikravindrae by the reduction of conidiophores to conidiogenous cells, cylindrical to ampulliform, pale brown conidiogenous cells with pointed, hyaline apex and brown to dark brown, smooth-walled conidia with dark equatorial slit. Additionally, A. acutiapicum is distinct from A. rasikravindrae by the reduction of conidiophores to pale brown conidiogenous cells and dimorphous, acropleurogenously arising conidia.

Culture Characteristics
Colonies grew on PDA at 20 • C in the dark attenuated 2 cm diam., within 7 days, flat, spreading, margin circular, with abundant aerial mycelia, surface and reverse white to off-white.

Notes
Arthrinium bambusae was introduced by Wang et al. (2018) from Guangdong Province, China, where our collection also was obtained. However, the exact locality is not mentioned in the original description there. The morphology of our collection was obtained from fungal structures on the host specimen, while Wang et al. (2018) had described the fungus from sporulated cultures. However, the morphology of our collection is similar to the holotype. Phylogenetically, A. bambusae clusters with A. garethjonesii, A. neogarethjonesii, A. mytilomorphum, A. setostromum, and A. subroseum with strong bootstrap value (ML/PP = 100/1), and the A. bambusae isolate (KUMCC 20-0207) clustered well with the ex-type culture (ML/PP = 96/1).

Culture Characteristics
Colonies grew on PDA at 20 • C in the dark attenuated 2 cm diam., within 5 days, flat, wooly, margin circular, with slight aerial mycelia, surface initially white, becoming grayish white and reverse yellowish white.

Culture Characteristics
Colonies grew on PDA at 20 • C in the dark attenuated 2 cm diam., within 5 days, flat, spreading, circular, margin filiform with abundant aerial mycelia, surface white to off-white and reverse pale yellow, sporulation occurs on 2% PDA incubated at 25 • C after 2 weeks, black, conidial mass concentrated at colony margins. Sporulation occurred after 10 days on PDA incubated at 20 • C in the dark without any host substrate. Conidia seem black mass and spread mostly in colony margins.
Additionally, A. pseudorasikravindrae shows close phylogenetic affinities to A. chinense, A. paraphaeospermum, and A. rasikravindrae (Figure 1). Arthrinium pseudorasikravindrae is morphologically distinct from the above species (Table 2) by its thick-walled, finely roughened conidia with pale, equatorial slit and ampulliform, cylindrical or doliiform, basauxic conidiogenous cells. The morphology of A. pseudorasikravindrae is compared with other closely related species (Table 2). Therefore, considering morphological and molecular uniqueness, these isolates are introduced here as belonging to a new species, A. pseudorasikravindrae. HKAS 107669 and HKAS 107670 represent a distinct clade (clade A, Figure 1) which not known before in the phylogenetic analysis, and hence, these collections are introduced here as a new species based on their morphology and phylogeny.

DISCUSSION
Bamboo is an important group of flowering plants that helps to conserve and manage forest ecosystems and reduce soil erosion and it is also important for panda conservation and many more commercial applications such as making fishing rod, flute, paper, flooring material, etc. and as food for humans and livestock (Chapman and Peat, 1992). Members of bamboo belong to the family Poaceae comprising more than 115 genera with approximately 1,450 species (Gratani et al., 2008;Kelchner and Group, 2013), and bamboo occurs in all tropical, subtropical, and temperate regions as herbaceous or woody plants. Microfungi associate with bamboo in many ways and phytopathogenic or endophytic microfungi form diseases while saprobic microfungi help to decompose plant debris (Zhang and Wang, 1999;Hyde et al., 2002a,b).
The first monograph on bambusicolous fungi was published with 258 fungal species by Hino and Katumoto (1960), and 63 new species were introduced by Petrini et al. (1989). Eriksson and Yue (1998) provided a checklist of the ascomycetes on bamboo, while Zhang and Wang (1999) recorded 213 species described from bamboo in China. Kuai (1996) listed phytopathogenic bambusicolous fungi in China and Taiwan. Hyde et al. (2002a) reviewed bambusicolous fungi that grow on all bamboo substrates including the leaves, culms, branches, rhizomes, and roots and enlisted more than 1,100 species, which belong to 228 genera. Dai et al. (2018) have reviewed the taxonomy of bambusicolous fungi. This study is one of the articles in the series on bambusicolous microfungi in Guangdong Province. Herein, we collected Arthrinium-like taxa from bamboo plant samples from Shenzhen, Guangdong Province, China. Currently, there are 81 species in the Arthrinium (Species Fungorum, 2020) and only 61 have molecular data. More than 30% of holotypes of Arthrinium species have been collected in China (Table 1). Therefore, the aims of this paper were to study Arthrinium-like fungi in Guangdong Province and to introduce several putative new species by comparing them morphologically and genetically with existing taxa.
According to morphology and phylogeny, two novel Arthrinium species were obtained with two new locality records. Most phylogenetic studies on Arthrinium used ITS, β-tubulin, and tef 1-α; however, LSU has been added to the analyses here. Negligible variations occur in tree topology in spite of adding LSU. A. guizhouense (HKAS 107672) is the first record in Guangdong Province and also from bamboo. The holotype of A. guizhouense was collected from the air in kart caves in Guizhou Province, China . This suggests that fungal conidioma in plant hosts release the conidia and conidia can survive in the air for a sufficiently long time. Our strain of A. bambusae is identical to the holotype which was collected from Guangdong Province on bamboo . Hence, this specimen can be used as an epitype if the holotype cannot be used for taxonomic purpose. The morphological differences between these two Arthrinium species are listed in Table 2. However, the life mode, host, and colony characters of these two species are not significantly different.

DATA AVAILABILITY STATEMENT
The datasets generated in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ supplementary material.

AUTHOR CONTRIBUTIONS
IS designed the study, performed the morphological study and phylogenetic analyses, and wrote the manuscript. JB, NX, and RC reviewed and edited the manuscript. All authors approved the final manuscript.