One New Species and Two New Host Records of Apiospora from Bamboo and Maize in Northern Thailand with Thirteen New Combinations

The genus Apiospora is known as a cosmopolitan genus, found across various substrates. In this study, four Apiospora taxa were obtained from the decaying stems of bamboo and maize in northern Thailand. Apiospora collections were compared with known species based on the morphological characteristics and the DNA sequence data of internal transcribed spacer (ITS), the partial large subunit nuclear rDNA (LSU), the translation elongation factor 1-alpha gene (TEF1-α) and beta-tubulins (TUB2). Apiospora chiangraiense sp. nov. and two new host records (Ap. intestini and Ap. rasikravindra) are introduced here based on the morphological characteristics and multi-locus analyses. Additionally, thirteen species previously identified as Arthrinium are introduced as new combinations in Apiospora, viz., Ap. acutiapica, Ap. bambusicola, Ap. biserialis, Ap. cordylines, Ap. cyclobalanopsidis, Ap. euphorbiae, Ap. gelatinosa, Ap. locuta-pollinis, Ap. minutispora, Ap. pseudorasikravindrae, Ap. septate, Ap. setariae and Ap. sorghi.


Introduction
Apiospora was introduced by Saccardo with Ap. montagnei as the type species [1]. The genus was reported in both sexual and asexual morphs. The sexual morphs are characterized by multi-locular perithecial stromata with hyaline ascospores surrounded by a thick gelatinous sheath [2][3][4]. The asexual morph of Apiospora was characterized by basauxic conidiogenesis, with globose to subglobose conidia, which are usually lenticular in the side view, obovoid and pale brown to brown [2,5,6]. Species of Apiospora are similar in morphology, thus it is difficult to distinguish them without molecular phylogenetic data. The size, color and shape of conidia and the morphology of conidiophores (e.g., size, shape and septation) should be used together to better identify them. For example, conidiophores of some species reduce to conidiogenous cells (e.g., Ap. bambusae, Ap. acutiapicum), while some species have semi-micronematous to macronematous conidiophores (e.g., Ap. bambusicola, Ap. intestini).
Apiospora species have a worldwide distribution and can be found from various hosts [3,[7][8][9]. Most Apiospora species are associated with plants as endophytes, pathogens Fresh specimens of bamboo and maize culms with fungal fruiting bodies were collected from Chiang Rai, Thailand from September-October 2020. Specimens were brought to the laboratory in plastic Ziploc bags for observation. Senanayake et al. [27] were followed for the morphological observations and single-spore isolation. The morphological characteristics were examined under a stereomicroscope (Motic SMZ-171, Wetzlar, Germany). The conidiomata were observed and photographed using a Nikon ECLIPSE Ni-U compound microscope connected to a Nikon camera series DS-Ri2 (New York, United States). The germinating ascospores were transferred aseptically to fresh potato dextrose agar (PDA) media and incubated at room temperature (25 • C) for 2-4 weeks. The morphological characteristics of cultures were checked and recorded after 30-60 days.
The herbarium specimens have been deposited at the herbarium of Mae Fah Luang University (MFLU) and Kunming Institute of Botany (HKAS), while the living cultures have been deposited at Mae Fah Luang University Culture Collection (MFLUCC). The Faces of Fungi and the Index Fungorum numbers are registered as outlined in Jayasiri et al. [28], and the Index Fungorum [26].

DNA Extraction, PCR Amplification and Sequencing
The genomic DNA was extracted from living pure cultures using the Biospin Fungus Genomic DNA extraction Kit (BioFlux, P.R. China) following the manufacturer's protocol. The internal transcribed spacer (ITS) with the primer pair of ITS4/ITS5 [29], the partial large subunit nuclear rDNA (LSU) with the primer pair of LR0R/LR5 [30], the translation elongation factor 1-alpha gene (TEF1-α) with the primers of EF1-728F/EF-2 [31,32] and the TUB2 with primers of bt2a/bt2b [33] were used to amplify the genes ITS, LSU, TEF1-α and TUB2. The polymerase chain reaction (PCR) was carried out under the following protocol: the final volume of 25 µL consisting of 2 µL of DNA template, 1 µL of each forward and reverse primers, 12.5 µL of 2× FastTaq Premix (mixture of Taq DNA polymerase, dNTPs, and a buffer) and 9.5 µL of deionized water. The PCR thermal cycle program was as follows: for ITS and LSU: initial denaturation at 95 • C for 5 min, then 35 cycles of denaturation at 94 • C for 30 s, annealing at 52 • C for 30 s and extension at 72 • C for 1 min and final extension at 72 • C for 10 min; for TEF1-α: initial denaturation at 94 • C for 5 min, then 35 cycles of denaturation at 94 • C for 1 min, annealing at 56 • C for 1 min and extension at 72 • C for 90 s and final extension at 72 • C for 10 min; for TUB2: initial denaturation at 95 • C for 5 min, then 35 cycles of denaturation at 94 • C for 45 s, annealing at 55 • C for 45 s and extension at 72 • C for 1 min and final extension at 72 • C for 10 min. The PCR products were checked in 1% agarose gels and sent to Tsing Ke Biological Technology (Kunming) Co., China for sequencing. The sequence quality was checked, and the sequences were condensed with SeqMan. The sequences derived in this study were deposited in the GenBank, and the accession numbers were obtained (Table 1).
The construction of the combined phylogenetic trees was completed using maximum likelihood (ML) and Bayesian inference posterior probabilities (BYPP), with Sporocadus trimorphus (CBS 114203) as the outgroup taxon. The models were selected as GTRGAMMA for maximum likelihood, while the best-fit models were selected as GTR + I + G for ITS, LSU and HKY + I + G for TUB2, and TEF1-α for the Bayesian posterior probability analysis. The maximum likelihood (ML) analysis was performed using RAxML-HPC v.8 [42,43] on the XSEDE TeraGrid of the CIPRES Science Gateway (https://www.phylo.org, accessed on 12 August 2021) [44] with a rapid bootstrap analysis, followed by 1000 bootstrap replicates. The final tree was selected amongst the suboptimal trees from each run by comparing the likelihood scores under the GTRGAMMA substitution model. The Bayesian analyses were performed by MrBayes v. 3.2 [45]. Markov chain Monte Carlo (MCMC) was run for 5,000,000 generations, and the trees were sampled every 100th generation. The first 10% of the trees that represented the burn-in phase were discarded, and only the remaining 90% of the trees were used for calculating the posterior probabilities (PP) for the majority rule consensus tree. Phylogenetic trees were visualized with FigTree v. 1.4.2 [46] and modified in Adobe Illustrator CS5 software (Adobe Systems, USA). The newly obtained sequences in this study were deposited in the GenBank.
Saprobic on dead culms of bamboo. Sexual morph: undetermined. Asexual morph: Colonies on natural substrate are dry, dark brown to black, velvety, dull, consisting of a sterile mycelial outer zone and a round, glistening, abundantly sporulating center, with conidia readily liberated when disturbed. Mycelium is superficial, branched, hyaline to
Apiospora rasikravindrae was originally isolated from soil in Norway [47]. Apiospora rasikravindrae occurred on Capsicum, Kappaphycus alvarezii, Pinus, Platanus acerifolia, rice, Sargassum thunbergia and Triticum aestivum from Brazil, China, India, Japan, Netherlands, Svalbard and Thailand [3,48]. Dai et al. [3] describe and illustrate both sexual and asexual morphs for Ap. rasikravindrae and it was collected on the stems of bamboo. In this study, the isolate MFLUCC 21-0051 was newly collected from bamboo, while the isolate MFLUCC 21-0054 was newly recorded from maize.
Notes: Arthrinium acutiapicum was introduced by Senanayake et al. [34] and was collected on dead twigs of Bambusa bambos in China. Senanayake et al. [34] mentioned that this species is distinct from Ar. pseudorasikravindrae, which is a sister to Ar. acutiapicum, by the reduction of conidiophores to conidiogenous cells, cylindrical to ampulliform, pale brown conidiogenous cells with pointed, hyaline apex and brown to dark brown and smooth-walled conidia with a dark equatorial slit [34].
In our phylogenetic analysis based on combined LSU, ITS, TEF1-α and TUB2 sequence data, Arthrinium acutiapicum clustered with Apiospora pseudorasikravindrae (=Ar. pseudorasikravindrae) with high support (ML/BI = 95/-). Thus, we propose the transfer of Ar. acutiapicum under the new combination Ap. acutiapica, based on the morphological and phylogenetic analyses. Notes: Arthrinium bambusicola was introduced by Tang et al. [18] and was collected on dead culms of Schizostachyum brachycladum in Thailand. Tang et al. [18] mentioned that Ar. bambusicola were retrieved as a sister taxon of Ar. gutiae with high support (ML/BI = 83/0.99), but differs from Ar. gutiae in having larger conidia and irregularly rounded, guttulate to roughened conidia. Pintos and Alvarado [4] transferred Ar. gutiae to Apiospora based on the phylogenetic analyses and morphological characters.
In our phylogenetic analyses based on combined LSU, ITS, TEF1-α and TUB2 sequence data, Arthrinium bambusicola is a sister to the newly introduced species Ap. chiangraiense with high support (ML/BI = 80/0.99). Thus, we propose the transfer of Ar. bambusicola under the new combination Ap. bambusicola, based on the morphological and phylogenetic analyses.
In our phylogenetic analyses, Arthrinium gelatinosum clustered with Apiospora biserialis with high support (ML/BI = 90/0.99). Thus, we propose the transfer of Ar. gelatinosum under the new combination Ap. gelatinosa. Notes: Arthrinium pseudomarii was introduced by Chen et al. [39] from the leaves of Aristolochia debilis in China. Chen et al. [39] mentioned that Ar. pseudomarii differs from Ar. hispanicum, Ar. marii and Ar. mediterranei by larger, subglobose to ellipsoid conidia and showed a close relationship with three species with high bootstrap support values (ML/MP = 95/93) [39].
In our phylogenetic analyses, Ar. pseudomarii (GUCC 10228) is a sister to Ap. locutapollinis (=Ar. locuta-pollinis) with high support of 95% ML. Based on the nucleotide comparisons, Ar. pseudomarii is slightly different from Ap. locuta-pollinis in 10/582 bp (1.72%) of ITS, but no base pair differences were observed in TUB2 and TEF1-α. Morphologically, the conidia of Ar. pseudomarii are similar to the holotype Ap. locuta-pollinis (LC 11683) in having similar size, brown with a hyaline equatorial rim, smooth, subglobose to ellipsoid condia and hyaline to pale brown, smooth, ampulliform to doliiform conidiogenous cells aggregated into clusters on the hyphae. Thus, we identified that they are the same species, and we synonymize Ar. pseudomarii under the Ap. locuta-pollinis, based on the morphological and phylogenetic analyses. Notes: Arthrinium minutisporum was introduced by Das et al. [37] from mountain soil in Korea. Morphologically, Ar. minutisporum is quite similar to Ar. phragmites, Ar. aureum and Ar. Hydei. However, the conidia of Ar. minutisporum are smaller than those of Ar. phragmites, Ar. aureum and Ar. Hydei, and Ar. minutisporum produce smaller conidiogenous cells than Ar. phragmites [39]. Pintos and Alvarado [4] transferred Ar. phragmites, Ar. aureum and Ar. hydei to Apiospora phragmites, Ap. aureum and Ap. hydei, based on the phylogenetic analyses and morphological characteristics. Whereas Ar. minutisporum was maintained in Arthrinium.
In our phylogenetic analyses, Arthrinium minutisporum forms a distinct subclade and is close to Apiospora aurea, Ap. balearica and Ap. descalsii with strong bootstrap support values (ML/PP = 99/1.00) within Apiospora. Thus, we propose the transfer of Ar. minutisporum under the new combination Ap. minutispora.
Our phylogenetic analyses based on combined LSU, ITS, TEF1-α and TUB2 sequence data show Ar. pseudorasikravindrae is a sister to the new combinations Ap. acutiapica (=Ar. acutiapicum) with high support (ML/BI = 77/0.99). Thus, we propose the transfer of Ar. pseudorasikravindrae under the new combination Ap. pseudorasikravindrae. Notes: Arthrinium septatum was introduced by Feng et al. [49] from dead bamboo culms in China. Feng et al. [49] showed that Arthrinium septatum forms a well-supported clade and appears to be distinct from other Arthrinium species. Arthrinium septatum resembles Ar. biseriale in having a biseriate, broad fusiform to cylindrical ascospores and cylindrical, clavate asci. However, Ar. septatum differs from Ar. biseriale by having smaller stromata [49].
In our phylogenetic analyses, Arthrinium septatum groups in a well-supported clade with Ap. pseudospegazzinii and Ap. gelatinosa. Thus, we propose the transfer of Ar. septatum under the new combination Ap. septata, based on the morphological and phylogenetic analyses.  [38] from Setaria viridis in China. Jing et al. [38] mentioned that this species has larger conidia and is phylogenetically closely related to Ar. jiangxiense. Pintos and Alvarado [4] transferred Ar. jiangxiense to Apiospora and synonymized Ap. jiangxiens based on the phylogenetic analyses and morphological characteristics.
In our phylogenetic analyses based on combined LSU, ITS, TEF1-α and TUB2 sequence data, Arthrinium setariae clustered with Apiospora jiangxiense with high support (ML/BI = 87/1.00). Thus, we propose the transfer of Ar. setariae under the new combination Ap. setariae, based on the morphological and phylogenetic analyses. Notes: Arthrinium sorghi was introduced as an endophyte by Bezerra et al. [36] from the leaves of Sorghum bicolor in Brazil. Bezerra et al. [36] mentioned that Ar. sorghi is treated as a unique lineage within Arthrinium based on ITS phylogenetic analysis. Morphologically, Ar. sorghi resembles Ar. pseudosinense, Ar. ovatum and Ar. phaeospermum, but differs from them by the culture characteristics, conidiophores and conidia size [36]. Pintos and Alvarado [4] transferred Ar. pseudosinense, Ar. ovatum and Ar. phaeospermum to Apiospora pseudosinensis, Ap. ovata and Ap. phaeospermum based on the phylogenetic analyses and morphological characteristics.
In our phylogenetic analyses based on combined LSU, ITS, TEF1-α and TUB2 sequence data, Arthrinium sorghi clustered with Apiospora bambusucila with high support (ML/BI = 78/0.99). Thus, we propose the transfer of Ar. sorghi under the new combination Ap. sorghi, based on the morphological and phylogenetic analyses.

Discussion
In this study, the new taxon Apiospora chiangraiense and two new host records, viz., Ap. intestini and Ap. rasikravindrae, are introduced based on the morphological and phylogenetic analyses. In addition, thirteen new combinations are proposed based on the phylogenetic analyses.
Apiospora was previously synonymized under Arthrinium, but Pintos et al. [14] reevaluated the placements of these two genera and transferred 55 species to Apiospora based on a phylogenetic analysis. Currently, 117 species of Apiospora are listed in the Index Fungorum [33]. Among these 117 species, 55 species have been confirmed in Apiospora by phylogenetic analyses [4]; however, the remaining 62 species need to be confirmed, as the sequence data of these species are not available. The morphology of Apiospora and Arthrinium are quite similar, so it is difficult to distinguish Apiospora and Arthrinium based only on morphology.
In the phylogenetic analyses, two Arthrinium species, viz., Arthrinium trachycarpum and Ar. urticae, formed a distinct clade out of Arthrinium, and this result is consistent with previous studies [18]. However, the morphologies of these two species are not significantly different from Arthrinium; thus, more collections are required to clarify the placement of these two species [24,50]. In addition, our phylogenetic analyses showed that Apiospora sorghi, Ap. bambusucila, Ap. chiangraiense and Ap. intesini are not clustered together in Apiospora major clades ( Figure 1). We also compared the LSU sequence of these four species with other Apiospora species, but a few base pair differences were found. Moreover, their morphologies fit well within the species concept of Apiospora. Thus, further phylogenetic analyses are necessary to confirm whether Apiospora is a species complex or not.