Additions to the Genus Arthrinium (Apiosporaceae) From Bamboos in China

Arthrinium has a widespread distribution occurring in various substrates (e.g., air, soil debris, plants, lichens, marine algae and even human tissues). It is characterized by the basauxic conidiogenesis in the asexual morph, with apiospores in the sexual morph. In this study, seventeen isolates of Arthrinium were collected in China. Based on their morphology and phylogenetic characterization, four new species (A. biseriale, A. cyclobalanopsidis, A. gelatinosum, and A. septatum) are described and seven known species (A. arundinis, A. garethjonesii, A. guizhouense, A. hydei, A. neosubglobosa, A. phyllostachium and A. psedoparenchymaticum) are identified, of which the sexual morph of three species (A. guizhouense, A. phyllostachium and A. psedoparenchymaticum) and asexual morph of A. garethjonesii are reported for the first time. The detailed descriptions, illustrations and comparisons with related taxa of these new collections are provided. Phylogenetic analyses of combined ITS, LSU, TUB2, and TEF sequence data support their placements in the genus Arthrinium and justify the new species establishments and identifications of known species.

we are carrying out the survey of fungal diversity in Karst formations of the Asian region, and many new taxa are described in last few years (Li et al., 2016;Chen et al., 2017Chen et al., , 2020Zhang et al., 2017Zhang et al., , 2019Feng et al., 2019;Liu et al., 2019;Dissanayake et al., 2020b). In this study, seventeen Arthrinium-like were collected in Guizhou and Guangdong province, China and can be recognized as eleven Arthrinium species based on morphological characters and phylogeny inferred from the multi-gene sequences data (ITS, LSU, TUB2, and TEF) analyses, which four new species (A. biseriale, A. cyclobalanopsidis, A. gelatinosum, and A. septatum) and seven known species (A. arundinis, A. garethjonesii, A. guizhouense, A. hydei, A. neosubglobosa, A. phyllostachium and A. psedoparenchymaticum) are introduced and identified, respectively. The aim of this study is to describe these new taxa with detailed descriptions and illustrations, and also provide their phylogenetic relationships within Arthrinium based on multi-gene analysis.

Sample Collection, Morphological Studies and Isolation
Samples were collected from Guizhou and Guangdong Province in China. Fungal fruiting bodies were examined by using stereomicroscope (Motic SMZ 168). Free hand sections of fungal structures were mounted in water for microscopic studies and photomicrography. Images were taken by using a Nikon ECLIPSE Ni compound microscope fitted with a Canon EOS 70D digital camera. All measurements were taken by using Tarosoft Image Frame Work software (IFW) (Liu et al., 2010), and photo plates were processed with Adobe Photoshop CS6 software (Adobe Systems, United States).
The single spore isolation followed the method described in Senanayake et al. (2020b). Parts of morphological descriptions were based on sporulated cultures on WA (Water Agar) at room temperature (ca. 25 • C). Type specimens were deposited in the herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (HKAS), Kunming, China and Guizhou Academy of Agriculture sciences Herbarium (GZAAS). Pure cultures were deposited in China General Microbiological Culture Collection Center (CGMCC) and Guizhou Culture Collection (GZCC). Faces of Fungi 1 number is obtained as described in the paper by Jayasiri et al. (2015), and the new taxa are registered in Index Fungorum (2021).

DNA Extraction, PCR Amplification and Sequencing
Fungal mycelia were scraped from the pure culture which were growing on PDA (Potato Dextrose Agar) for one week at 25 • C in dark. DNA was extracted by using Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech, China) from fresh fungal mycelia, but some were extracted directly from fruiting bodies by using Forensic DNA Kit (Omega Bio-Tek, China). Four gene regions, large subunit rDNA (LSU), internal transcribed spacer (ITS), beta-tubulin (TUB2) and the translation elongation factor 1-alpha (TEF) gene were amplified by the primer pairs LR0R and LR5 (Vilgalys andHester, 1990), ITS5 andITS4, T1 (O'Donnell andCigelnik, 1997) and Bt2b (Glass and Donaldson, 1995), EF1-728F and EF-2 (O'Donnell et al., 1998;Carbone and Kohn, 1999), respectively. Polymerase chain reaction (PCR) was carried out in 25 µL reaction volume containing 12.5 µL 2 × PCR Master Mix (Sangon Biotech, China), 9.5 µL ddH 2 O, 1 µL of each primer and 1 µL DNA template. The annealing temperatures were adjusted to 56 • C for ITS, LSU and TUB2, and 55 • C for TEF. PCR products were sent to sequence at Sangon Biotech Co., Ltd., China. The PCR products were examined using 1.2% agarose electrophoresis gel stained with ethidium bromide. PCR products were purified and sequenced by Sangon Biotech (Shanghai) Co., Ltd., China. New generated nucleotide sequences were submitted in GenBank (Table 1).

Phylogeny
To determine the phylogenetic placement of the new collections in this study, the combined ITS, LSU, TUB2 and TEF data set comprised 119 taxa with Seiridium phylicae (CPC 19962 and CPC 19965) as the outgroup taxa. The concatenated alignment comprises 2,770 characters (ITS: 1-635; LSU: 636-1,454; TUB2: 1,455-2,300; TEF: 2,301-2,770) including gaps, of which 1,361 characters were constant, and 1,279 characters are parsimony informative and 130 are parsimony uninformative. Maximum    A. serenense The newly generated sequence is shown in bold.  The gamma distribution shape parameter alpha is equal to 0.332641 and the Tree-Length equal to 3.795226. Phylogenetic analyses showed that our newly collected seventeen taxa clustered into eleven clades and can be recognized as seven known species (Arthrinium arundinis, A. garethjonesii, A. guizhouense, A. hydei, A. neosubglobosa, A. phyllostachium, A. psedoparenchymaticum) and four new species (A. biseriale, A. cyclobalanopsidis, A. gelatinosum, A. septatum) (Figure 1).

Taxonomy
Arthrinium biseriale Y. Feng and Z.Y. Liu, sp. nov. Figure 2 Index Fungorum number: IF558136 Facesoffungi number: FoF 09569 Etymology: The epithet refers to the ascospores are arranged in two rows in the ascus.

Culture characters
Ascospores germinated on WA within 24 h and germ tubes produced from middle and lower end. Colonies fast The tree is rooted with Seiridium phylicae (CPC 19962 and CPC 19965). New strains are shown in red.

Culture characteristics
Ascospores germinated on WA within 24 h. Colonies on PDA reached 8 cm in 7 days at 25 • C, flat, aerial mycelium white. The hyphae in the center are cottony, dense, and there is a thin circle of hyphae at the edge. Reverse grayish white with a dirty white patch.

Culture characteristics
On PDA, colonies very fast, reached 8 cm in 8 days at 25 • C, velvety, circular, with regular edge, middle densely and raised white, dense at above the from margin, aerial mycelia, surface initially white, became grayish and reverse white.

Notes
Arthrinium guizhouense was introduced from air in a karst cave (asexual morph was provided from the culture) in Guizhou province, China by Wang et al. (2018). In this study, one collection was found as saprobe on bamboo in Guangzhou, China and it is identified as A. guizhouense based on the phylogeny and morphology evidences. In addition, our new collection provides the sexual morph which only the asexual morph was illustrated by Wang et al. (2018) and Senanayake et al. (2020a).

Culture characters
Ascospores germinated on WA within 24 h and germ tubes produced from Upper, middle and lower end. Colonies fast growed on PDA at 25 • C, flat, spreading, the middle is white, smooth, the edge surface tapetum, gray-white, indistinct aerial hyphae, the reverse side is yellowish in the middle, and the edge is grayish white.

Notes
Arthrinium phyllostachium was introduced by Yang et al. (2019) based on the asexual morph and phylogeny analyses. It was collected from culms of Phyllostachys heteroclada (Poaceae) in China . The phylogenetic results showed that our new collections are identical to A. phyllostachium, Yang et al. (2019) only provided the asexual morph and the sexual morph is given in this study.

Culture characteristics
Ascospores germinating on WA within 24 h and germ tubes produced from upper. Colonies fast growing on PDA at 25 • C, under 12 h light/12 h dark, cottony, circular, sparse, raised, with irregular edge, white in center.

Notes
Arthrinium pseudoparenchymaticum was introduced by Wang et al. (2018) based on the asexual morph characters and phylogeny analyses. It was originally collected from bamboo in China (Wang et al., 2018). In this study, a fresh specimen was collected and it is identical to A. pseudoparenchymaticum (Figure 1), both sexual and asexual morphs were described and illustrated (Figure 9).

Culture characteristics
The colony is flat, cotton-like, thick and dense, with sparse aerial mycelia. The surface of PDA is white and the reverse side is grayish white.

Notes
Our collection clusters together with the isolates of Arthrinium arundinis (Figure 1) and its morphology lines up with the type species. Therefore we identify it as Arthrinium arundinis.

Notes
This collection is identified as Arthrinium hydei based on both morphological characters and molecular data. Crous and Groenewald (2013) originally described A. hydei based on the asexual morph from a culture (CBS 114990) which was isolated from bamboo culms in Hong Kong, China; we found this species in Guizhou from the substrate in nature with its asexual morph.

Notes
Arthrinium neosubglobosa was introduced by Dai et al. (2016) based on the sexual morph characters and phylogeny analyses. The pure culture was attempted by single spore isolation and the DNA was extracted directly from the fruiting body, the new collection is identified as A. neosubglobosa based on the phylogeny (Figure 1) and morphology evidences.

DISCUSSION
Arthrinium species have been reported from many hosts, includes hive-stored pollen lichens, marine algae, soil debris, gut of insects and nodules of human skin (Sharma et al., 2014;Crous et al., 2015, Senanayake et al., 2015Wijayawardene et al., 2017, Zhao et al., 2018, it can be concluded that Arthrinium is ecologically diverse. Bamboo, as one of the most reported host, is a gramineous plant integrating economy and ornamental value (Gratani et al., 2008;Kelchner and Bamboo Phylogeny Group, 2013;Dai et al., 2016Dai et al., , 2017Jiang et al., 2018Jiang et al., , 2019Jiang et al., , 2020Wang et al., 2018;Yang et al., 2019), there is more than 115 genera with approximately 1,450 species. According to incomplete statistics, more than 1,100 species of fungi on bamboo were reported (Hyde et al., 2002a,b;Dai et al., 2016Dai et al., , 2017Senanayake et al., 2020a;Tang et al., 2020;Wijesinghe et al., 2020). It is of great significance to excavate and identify the fungi on bamboo.
Several studies have shown (Crous and Groenewald, 2013;Dai et al., 2016, Dai et al., 2017Wang et al., 2018;Yang et al., 2019) that it is difficult to identify the Arthrinium species solely rely on morphology and the multi-gene phylogenetic analyses are needed in the identification and classification of Arthrinium. The morphology of conidia is variable which can be depending on the period of incubation on different habitats, for example, A. biseriale, A. gelatinosum and A. septatum, are very similar in morphology, but molecular data distinguish them into different species; Our collection A. pseudoparenchymaticum differs from the type specimen (LC 8173) in the morphology of conidiophores, the size of conidiogenous cells is also different, while the molecular data supported them as the same species. These results are in agreement with the previous observations and publications (Crous and Groenewald, 2013;Dai et al., 2016, Dai et al., 2017Wang et al., 2018. In addition, as a high diverse group, it is also difficult to distinguish species within Arthrinium by only using ITS and LSU gene regions, and the protein genes (TEF and TUB2) are not available for many species in the genus which bring potential problem once the new or existing taxa are introduced and identified, respectively. For example the absent of the protein genes (TEF and TUB2) of Arthrinium garethjonesii would bring the troubles in identification of Arthrinium setostromum as it is hard to confirm whether they are same species or not as they are identical in ITS and LSU gene regions, as well as the close phylogenetic relationship (Figure 1). It would be necessary to provide protein genes when new taxa are introduced in these well-study and diverse groups.

DATA AVAILABILITY STATEMENT
The data presented in the study can be found in the Genbank. The accession numbers of the sequences deposited in GenBank are ITS: MW481705-MW481721; LSU: MW478885-MW478901; TEF:MW522938-MW522954; and TUB: MW522955-MW522969.