﻿Morphological and phylogenetic analyses reveal two new species and a new record of Apiospora (Amphisphaeriales, Apiosporaceae) in China

﻿Abstract The genus Apiospora includes endophytes, pathogens and saprobes, with a wide host range and geographic distribution. In this paper, six Apiospora strains isolated from diseased and healthy tissues of bamboo leaves from Hainan and Shandong provinces in China were classified using a multi-locus phylogeny based on a combined dataset of ITS, LSU, tef1 and tub2, in conjunction with morphological characters, host association and ecological distribution. Two new species, Apiosporadongyingensis and A.hainanensis, and a new record of A.pseudosinensis in China, are described based on their distinct phylogenetic relationships and morphological analyses. Illustrations and descriptions of the three taxa are provided, along with comparisons with closely related taxa in the genus.


Introduction
Apiospora Sacc., the type genus of Apiosporaceae K.D. Hyde, J. Fröhl., Joanne E. Taylor & M.E. Barr, was introduced by Saccardo with A. montagnei Sacc. as the type species (Saccardo 1875). The sexual morphs of Apiospora are characterized by multi-locular perithecial stromata with hyaline ascospores surrounded by a thick gelatinous sheath (Dai et al. , 2017Pintos and Alvarado 2021). The asexual morphs of Apiospora are characterized by their basauxic conidiogenesis, and globose to subglobose conidia, which are usually lenticular or obovoid in the side view, and pale brown to brown in color (Kunze 1817;Hyde et al. 1998;Dai et al. 2016). Most species of Apiospora are quite similar to each other in morphology, thus it is difficult to distinguish them without molecular phylogenetic data.
Until the studies of Pintos and Alvarado (2021) and Jiang et al. (2022a), the closely related genera Apiospora, Arthrinium Kunze and Neoarthrinium Ning Jiang were considered a single taxon because of their similar morphological characteristics, especially the basauxic conidiogenesis. However, the conidia of Apiospora and Neoarthrinium are more or less rounded in the face view and lenticular in the side view, whereas the conidia of Arthrinium are variously shaped (angular, curved, fusiform, globose, polygonal, navicular). In addition, the conidiophores of several Arthrinium and Neoarthrinium species have thick blackish septa, which are rarely observed in Apiospora (Pintos and Alvarado 2021;Tian et al. 2021;Jiang et al. 2022a). Apiospora species have a worldwide distribution and can be found on various hosts, while Arthrinium species are rarely found in tropical and subtropical habitats and commonly occur on Cyperaceae Juss. and Juncaceae Juss. (Ramos et al. 2010;Dai et al. 2017;Wang et al. 2018;Hyde et al. 2020;Pintos and Alvarado 2021;Tian et al. 2021). Four Neoarthrinium species have been discovered on four hosts from three distantly related host plant families in China, Colombia and Great Britain (Jiang et al. 2022a). Most Apiospora species are associated with plants as endophytes, pathogens or saprobes (Agut and Calvo 2004;Dai et al. 2016Dai et al. , 2017Tian et al. 2021). Some species are economically important plant pathogens, for example, A. arundinis causes bamboo brown culm streak, chestnut leaf spot and barley kernel blight (Martínez-Cano et al. 1992;Chen et al. 2014;Jiang et al. 2021), while A. sacchari causes damping-off of durum wheat (Mavragani et al. 2007). Some species have also been isolated from lichens, air, soil, seaweeds and animal tissues, and a few species are human pathogens which can cause cutaneous infections .
The aim of this study was to explore the diversity of Apiospora species in symptomatic and asymptomatic bamboo leaves collected in Hainan and Shandong provinces (China). We describe two newly discovered species, Apiospora dongyingensis and A. hainanensis, and a new record of A. pseudosinensis in China based on phylogenetic data and morphology.

Isolation and morphological studies
The samples were collected at the Diaoluoshan National Nature Reserve, Hainan Province, and the Dongying Botanical Garden, Shandong Province (China). The strains of Apiospora were isolated from single spores and fungal tissue obtained from diseased and healthy bamboo leaves following the methods described by Chomnunti et al. (2014). Sampled spores were suspended in sterile distilled water, spread onto potato dextrose agar (PDA) plates, and incubated for one day at 25 °C. After germination, the spores were transferred to a new PDA plate to obtain a pure culture. Additionally, about 25 mm 2 tissue fragments were taken from the margin of leaf lesions and their surface sterilized by consecutive immersions in a 75% ethanol solution for 60 s, 5% sodium hypochlorite solution for 30 s, and then rinsed in sterile distilled water for 60 s (Mu et al. 2021). The surface sterilized plant tissue was dried with sterilized paper and moved on the PDA plates. All the PDA plates were incubated at 25 °C for 3-4 days in darkness, and then hyphae were picked out of the periphery of the colonies and grown on new PDA plates (Jiang et al. 2022b).
After 7 days of incubation, the morphological characters of the colonies were recorded on PDA with a digital camera (Canon G7X). Morphological descriptions were based on cultures sporulating on water agar (WA). The size of the conidiogenous cells and conidia were shown as minimum-maximum. Color notations were done using the color charts of Rayner (1970). The micro-morphological characters of the colonies were studied using a stereomicroscope (Olympus SZX10) and a microscope (Olympus BX53), both fitted with high-definition color digital cameras. Grown cultures of Apiospora were stored in 10% sterilized glycerin and sterile water at 4 °C for further studies in the future. All specimens were deposited in the Herbarium of the Department of Plant Pathology, Shandong Agricultural University (HSAUP). Living cultures were deposited in the Shandong Agricultural University Culture Collection (SAUCC). Taxonomic information on the new taxa was submitted to MycoBank (http://www.mycobank.org).

DNA extraction and amplification
Genomic DNA was extracted from fungal mycelia grown on PDA, using a modified cetyltrimethylammonium bromide (CTAB) protocol as described in Guo et al. (2000). DNA sequences of four different loci were obtained, including the nrDNA internal transcribed spacer regions 1 and 2 with the intervening 5.8S subunit (ITS), a partial sequence of the large subunit nrDNA subunit (LSU), a partial sequence of the translation elongation factor 1-alpha gene (tef1), and a partial sequence of the beta-tubulin gene (tub2). They were all amplified with the primer pairs and polymerase chain reaction (PCR) program listed in Table 1. PCR was performed using an Eppendorf Master Thermocycler (Hamburg, Germany). Amplification reactions contained 12.5 μL 2× Taq Plus Master Mix II (Vazyme, Nanjing, China), 1 μL of each forward and reverse primers (10 μM) (Tsingke, Qingdao, China), 1 μL of template genomic DNA, and distilled deionized water to a total volume of 25 μL. The PCR products were visualized on 1% agarose electrophoresis gels. Bi-directional sequencing was conducted by the Tsingke Company Limited (Qingdao, China). Consensus sequences were obtained using MEGA 7.0 (Kumar et al. 2016). All sequences generated in this study were deposited in GenBank (Table 2).

Phylogenetic analyses
Newly generated ITS, LSU, tef1 and tub2 sequences from the six strains studied were aligned with all reference sequences of Apiospora and related species available in GenBank using the MAFFT v.7.11 online software (http://mafft.cbrc.jp/alignment/ server/, Katoh et al. 2019) with the default settings, manually correcting the resulting alignment where necessary. Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic analyses were conducted individually on each locus (ITS, LSU, tef1 and tub2) and on a combined dataset including all of them. The best-fitting evolutionary model of each partition was determined using MrModeltest v. 2.3 (Nylander 2004). ML and BI were run on the CIPRES Science Gateway portal (https://www. phylo.org/) using RaxML-HPC2 on XSEDE (8.2.12) (Miller et al. 2012;Stamatakis 2014) and MrBayes on XSEDE (3.2.7a), respectively (Huelsenbeck and Ronquist 2001;Ronquist and Huelsenbeck 2003;Ronquist et al. 2012). For ML analyses the default parameters were used, while BI was carried out using a Markov chain Monte Carlo (MCMC) algorithm. BI analyses included four MCMC chains and were run for 5,000,000 generations until the average standard deviation of split frequencies was below 0.01 with trees saved every 1000 generations. The burn-in fraction was set to 0.25 and posterior probabilities (PP) were determined from the remaining trees. The resulting 50% majority-rule tree was plotted using FigTree v. 1.4.4 (http://tree.bio. ed.ac.uk/software/figtree) and edited with Adobe Illustrator CS6.0.

Phylogenetic analyses
Among the six strains of Apiospora isolated from the samples studied, two new species were discovered, and another one found for the first time in China after the combined analysis of ITS, LSU, tef1 and tub2 DNA sequences from 89 isolates of Apiospora plus Arthrinium caricicola Kunze & J.C. Schmidt (CBS 145127) as the outgroup taxon. A total of 2241 characters including gaps were compared in the phylogenetic analysis, viz. ITS: 1-706, LSU: 707-1513LSU: 707- , tef1: 1514LSU: 707- -1932LSU: 707- , tub2: 1933LSU: 707- -2241. Of these characters, 1436 were constant, 271 were variable and parsimony-uninformative, and 534 were parsimony-informative. For the BI and ML analyses, the substitution model GTR+I+G was selected for all partitions.
The BI analysis reached the established convergence after 3935000 generations, resulting in 39351 sampled trees, of which 29514 trees were used to calculate the posterior probabilities. The ML tree topology agreed with that obtained from the BI analysis, and therefore, only one tree (the ML) is presented (Fig. 1). The four strains (SAUCC 0302, SAUCC 0303, SAUCC 1681 and SAUCC 1682) studied in the present work represent two independent clades, interpreted as newly discovered independent species. These are described below and accommodated under the new names Apiospora dongyingensis and  Type. China, Shandong Province: Dongying Botanical Garden, on diseased leaves of bamboo, 13 July 2022, R.Y. Liu, holotype HSAUP 0302, ex-type living culture SAUCC 0302.
Culture characteristics. Colonies on PDA flat with entire margin, aerial mycelium white to gray, floccose cottony; surface and reverse gray in the center and grayish margin. PDA attaining 78.5-86.5 mm in diameter after 7 days at 25 °C, growth rate 11.0-12.5 mm/day. Additional specimen examined. China, Shandong Province: Dongying Botanical Garden, on diseased leaves of bamboo, 13 July 2022, R.Y. Liu, paratype HSAUP 0303, ex-paratype living culture SAUCC 0303.
Culture characteristics. Colonies on PDA flat with irregular margin, aerial mycelium white to pale yellow, floccose cottony; reverse pale yellow to yellow. PDA attaining 69.5-78.5 mm in diameter after 7 days at 25 °C, growth rate 9.5-11.5 mm/day. Notes. Apiospora pseudosinensis was originally described from bamboo leaves collected in the Utrecht Botanical Garden of the Netherlands (Crous and Groenewald 2013;Pintos and Alvarado 2021). In the present study, DNA sequences obtained from two strains (SAUCC 0221 and SAUCC 0222) collected also from bamboo leaves, were not significantly different from those of A. pseudosinensis (Fig. 1). Morphologically, our strains were similar to the original description (conidia 8-10 × 7-10 μm diam. in surface view, 7-8 μm diam. in side view). We therefore consider the newly found strains as A. pseudosinensis (Crous and Groenewald 2013; Pintos and Alvarado 2021).

Discussion
The family Apiosporaceae was proposed to accommodate genera with apiosporous hyaline ascospores and a basauxic, Arthrinium-like conidiogenesis (Hyde et al. 1998). Crous and Groenewald (2013) synonymized Apiospora with Arthrinium on the basis of the one fungus-one name policy (Hawksworth et al. 2011). Crous and Groenewald (2013) also resolved the genetic identity of multiple species of Arthrinium (= Apiospora), analysing ex-type collections, and confirmed that most species occur in Poaceae (R.Br.) Barnh. hosts, although some were known from many other plant host families. However, with the aid of additional genetic data from the type species of Arthrinium, Ar. caricicola, Apiospora and Arthrinium were separated again as two distinct genera (Pintos and Alvarado 2021). Arthrinium species have variously shaped conidia and inhabit Cyperaceae and Juncaceae in temperate, cold or alpine habitats. Most Apiospora species have rounded/lenticular conidia and inhabit mainly Poaceae (and many other host plant families) in a wide range of habitats, including tropical and subtropical regions (Pintos and Alvarado 2021;Samarakoon et al. 2022). An epitype for the type species of Apiospora, A. montagnei, was recently proposed by Pintos and Alvarado (2022).
There are many Apiospora species found on bamboos across the world (Table 2). Bamboos (Poaceae) are distributed in tropical and subtropical to mild temperate regions, with the heaviest concentration and largest number of species in China. Due to their abundance and economic importance, it is of great significance to study and identify the fungi growing on bamboo (Feng et al. 2021). In the present study, two new species (Apiospora dongyingensis and A. hainanensis) are introduced, and another one (A. pseudosinensis) is reported for the first time in China. All of them were collected from bamboo leaves and described based on their phylogenetic data and morphological characters. The descriptions and molecular data for species of Apiospora represent an important resource for understanding the diversity of bamboo fungi.