Insight into the Taxonomic Resolution of Apiospora: Introducing Novel Species and Records from Bamboo in China and Thailand

Abstract

Taxonomic studies of bambusicolous fungi in China and Thailand have resulted in the collection of three fascinating saprobic coelomycetes strains. Morphology coupled with combined gene analysis of ITS, LSU, TUB2, and TEF1-α DNA sequence data showed that they belong to the genus Apiospora, family Apiosporaceae. A new species from Thailand, Apiospora mukdahanensis, and new records of A. locuta-pollinis from China are herein described. In addition, based on both morphological data coupled with phylogenetics and nomenclatural analyses, A. mori is proposed as a new combination. Maximum likelihood, maximum parsimony and Bayesian analyses were performed to clarify the phylogenetic affinities of the species obtained in this study. Newly obtained strains are compared with morphologically- and phylogenetically-related taxa. The comprehensive descriptions, illustrations, and updated phylogeny are provided and discussed for intra-and intergeneric relationships within Apiospora species.

1. Introduction

Apiospora is a large genus in the family Apiosporaceae (Amphisphaeriales, Sordariomycetes, Ascomycota) [1,2], which is ecologically diverse and distributed worldwide [2,3,4,5]. Most species have been identified as saprobes and endophytes of a range of plant hosts, mainly occurring on the family Poaceae [2,3,4,5,6,7,8,9,10,11,12]. In addition, some species have been reported as plant pathogens. For instance, A. kogelbergensis causes the blight disease of Schizostachyum [13], A. sacchari causes the damping-off of durum wheat (Triticum durum) [14], and A. xenocordella causes fruit blight on pistachio (Pistacia vera) [15]. Apiospora shows a cosmopolitan distribution in diverse substrates, including air [4,16], soil [4,16,17,18], freshwater [19], marine environments [20,21,22,23,24,25], lichens [26], insect guts [27], and human tissues [3,28,29,30]. Interestingly, some species (e.g., A. arundinis, A. sacchari) have been reported as a source of useful bioactive compounds, such as antifungal agents and enzymes [21,22,31], possessing great potential for their commercial applications in the pharmaceutical industries.

Apiospora is classified by asexual morph characteristics that produce basauxic conidiophores and unicellular globose to obovoid conidia, usually rounded in face view and lenticular in side view, with a longitudinal germ slit [2,7,10,11,32]. The sexual morph is characterized as having multi-locular perithecial stromata, clavate to broadly cylindrical asci and hyaline ascospores surrounded by a thick gelatinous sheath [2,7,8,11]. Apiospora was previously known as the sexual morph of the genus Arthrinium [33,34]. According to the International Code of Nomenclature for Algae, Fungi, and Plants (ICN) [35], Apiospora was a synonym of Arthrinium due to the early introduction of Arthinium and is more commonly used in the literature [3]. Crous and Groenewald [3] and Wang et al. [4] provided the upgraded phylogenetic trees of Arthrinium species (=Apiospora) using combined ITS, TEF1-α, and TUB2 sequence data with additional strains (collected from various hosts, substrates, and locations) and indicated that Arthrinium seems to be a species complex which needs further taxonomic revision and epitypification. Multi-gene phylogeny of ITS, LSU, TEF1-α, and TUB2 sequences conducted by Pintos et al. [5] revealed that Arthrinium caricicola, the type species, and other species of Arthrinium mostly found in Carex sp. formed independent lineages unrelated to other species of Arthrinium, and reported that Apiospora occurred on other hosts. However, the taxonomic placement of both genera was uncertain until Pintos and Alvarado [2] resolved this issue and presented Arthrinium and Apiospora as well-supported distinct clades suggesting they are separate genera.

The morphological identification of Apiospora species is challenging because most species share similar morphological characteristics (e.g., conidia). In addition, their morphological features can vary depending on incubation periods and different substrates [3]. Thus, morphological characteristics integrated with molecular phylogeny have been widely accepted to distinguish Apiospora species [3,4,5,6,7,8,9,12,17,36,37,38,39]. Presently, 117 epithets are recognized in Apiospora [40], comprising 76 Arthrinium species, which were synonymized under Apiospora [2,12,25]. The taxonomic position of other taxa, which lack sequencing information and comprehensive morphological descriptions, remain uncertain and require further study. In this study, we isolated apiospora-like taxa from bamboo in China and Thailand. The morphological characteristics and molecular analyses of ITS, LSU, TUB2, and TEF1-α were applied to determine a new species, Apiospora mukdahanensis, and one new record of A. locuta-pollinis. Furthermore, Arthrinium mori is also transferred to Apiospora as a new combination on the basis of phylogenetic evidence. The host association, geographical distribution, and species diversity of Apiospora are also discussed.

2. Materials and Methods

2.1. Sample Collection, Fungal Isolation and Morphological Examination

Dead and decaying bamboo specimens were collected during a series of field trips conducted in China and Thailand from the year 2019–2021. The specimens were packed in zip-lock plastic bags prior for further study. Fungal colonies on the host substrate were observed using a stereo microscope (Nikon SMZ800N, Tokyo, Japan). The micromorphological characteristics were documented and photographed with a compound microscope (Nikon Eclipse Ni U, Tokyo, Japan) equipped with Nikon DS-Ri2 camera. The measurements of fungal structures (i.e., conidiomata, conidiophore mother cells, conidiophores, conidiogenous cells, and conidia) were made using the Tarosoft (R) Image Frame Work program. Images used for figures were combined and edited using Adobe Photoshop CS6 Extended version 10.0 software (Adobe Systems, San Jose, CA, USA). Single-spore isolation was conducted to isolate the fungus as detailed in Senanayake et al. [41]. The germinating conidia were inoculated on potato dextrose agar (PDA) and incubated at 28 °C for two weeks. Culture characteristics were observed and described after four weeks. Axenic cultures were kept in 2 mL sterilized screw-cap tubes containing PDA for short-term storage and duplicated in 10% glycerol for long-term storage. The type specimen and living culture of Apiospora mukdahanensis were deposited in the herbarium of Mae Fah Luang University (MFLU) and the Mae Fah Luang University Culture Collection (MFLUCC), respectively. The specimens of A. locuta-pollinis and cultures were deposited in the Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (KUN-HKAS), and the Kunming Institute of Botany Culture Collection, Kunming, China (KUNCC), respectively. Index Fungorum numbers and Faces of Fungi numbers were obtained for the new taxa as detailed in Index Fungorum [42] and Jayasiri et al. [43], respectively.

2.2. DNA Extraction, PCR Amplification and Sequencing

The total genomic DNA was extracted from mature mycelium grown on PDA at 28 °C for two weeks using a Biospin Fungus Genomic DNA Extraction Kit (BioFlux^®, Hangzhou, China). Polymerase chain reaction (PCR) amplification was applied to amplify DNA fragments with three phylogenetic markers, including the internal transcribed spacers region of ribosomal DNA (ITS; ITS1-5.8s-ITS2) using primers ITS5 and ITS4 [44]; the partial 28S large subunit nuclear ribosomal DNA (LSU) using primers LR0R and LR5 [45]; and the translation elongation factor 1-α (TEF1-α) using primers EF1-728F and EF-2 [46,47]. The PCR reaction was carried out in the final volume of 25 μL, containing 2 μL template DNA (50 ng/μL), 12.5 μL of PCR Master Mix (0.5 mM of each primer, 50 U Taq DNA polymerase 400 mM of each dNTP, and 3 mM MgCl₂), 1 μL of each forward and reward primer and 8.5 μL of the sterilized double-distilled water (ddH₂O). The PCR thermal cycling programs for ITS, LSU and TEF1-α were adjusted by an initial denaturation at 94 °C for 3 min, followed by 40 cycles of denaturation at 94 °C for 45 s, annealing at 56 °C for 50 s, extension at 72 °C for 1 min and a final extension step at 72 °C for 10 min. The PCR products were processed for purification and sequencing by TsingKe Biological Technology, Kunming City, Yunnan Province, China. Newly generated sequences in this study were deposited in Genbank (Table 1).

2.3. Phylogenetic Analyses

The sequences generated by this study were supplemented with the related taxa resulting from the nucleotide blast search in GenBank (www.ncbi.nlm.nih.gov/blast/, accessed on 1 September 2022) and recent publications [2,12,25,39,48,49] (Table 1). The matrix of consensus sequences was aligned using MAFFT v. 7.475 on the web portal (http://mafft.cbrc.jp/alignment/server/index.html) [50] and then the ambiguous sites were manually trimmed using BioEdit 7.1.3.0 [51]. Phylogenetic trees based on the concatenated ITS, LSU, TUB2, and TEF1-α sequence data (analysis 1) and ITS, LSU, and TEF1-α sequence data (analysis 2) were inferred to clarify the phylogenetic relationships of Apiospora species using maximum likelihood (ML) and Bayesian inference (BI) analyses. In order to clarify the phylogenetic placements of new strains and related strains in A. locuta-pollinis/marii clade, ML, maximum parsimony (MP), and BI were analyzed based on the concatenated ITS, LSU, TUB2, and TEF1-α sequence data (analysis 3). Phylogenetic trees of these combined gene datasets were further compared to check the congruence of the tree topologies.

ML analyses were implemented using RAxML-HPC2 (v.8.2.12) on the CIPRES web portal (http://www.phylo.org/portal2/) [52]. The GTRGAMMAI model of nucleotide substitution with 1000 rapid bootstrap replicates was used. BI analyses were performed using MrBayes v.3.2.7a via the CIPRES web portal (http://www.phylo.org/portal2/). The optimal substitution model of nucleotide evolution was determined using MrModeltest v. 2.3 [53]. In the first and second analyses, GTR + I + G was chosen as the best-fit model for the ITS, LSU, and TEF1-α datasets, and HKY + I + G for the TUB2 dataset. For the third analysis, the best-fit model for the ITS, LSU, TUB2, and TEF1-α datasets were as follows: SYM + I, GTR, GTR + G, and K80 + I. Ten million Markov chain Monte Carlo sampling (MCMC) generations were run with six simultaneous Markov chains to calculate Bayesian posterior probabilities [54,55,56]. Trees were sampled every 100th generation. When the average standard deviation of split frequencies was constantly below 0.01, the runs were automatically stopped and the first 25% of the generated trees were discarded. The remaining trees were used to evaluate the posterior probabilities (PP) of the majority rule consensus tree. MP analyses were conducted with the heuristic search option, as implemented in PAUP v. 4.0b10 [57]. Clade stability was determined using a bootstrap analysis with 1000 replicates, random sequence additions with maxtrees set to 1000 [58]. The MP tree was described for descriptive tree statistics including Tree Length (TL), Consistency Index (CI), Retention Index (RI), Relative Consistency Index (RC), and Homoplasy Index (HI) under different optimality criteria. Phylogenetic trees were viewed in FigTree v1.4.0 [59] and formatted using Adobe Photoshop CS6 software (Adobe Systems, San Jose, CA, USA).

2.4. Host and Geographical Distribution of Apiospora Species

To investigate geographical distribution and host-substratum diversity of the Apiospora species, the data were summarized based on the USDA fungal database (https://nt.ars-grin.gov/fungaldatabases/, accessed on 1 September 2022), academic literature, and this study).

3. Results

3.1. Phylogenetic Analyses

Analysis 1: The combination of ITS, LSU, TUB2, and TEF1-α sequence dataset comprised 156 taxa of Apiospora, and other related taxa in the family Apiosporaceae. Sporocadus trimorphus (CBS 114203) was selected as the outgroup taxon (Figure 1). The final alignment consisted of 3264 total characters, including gaps (ITS: 1–641 bp, LSU: 642–1524 bp, TUB2: 1525–2366 bp, TEF1-α: 2367–3264 bp). The RAxML analysis of the integrated dataset yielded a best scoring tree with a final ML optimization likelihood value of −39,292.041642. The aligned sequence matrix contained 1874 distinct alignment patterns, with 38.58% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.237643, C = 0.255831, G = 0.254014, T = 0.252512; substitution rates AC = 1.214378, AG = 2.729624, AT = 1.181789, CG = 1.027511, CT = 4.357413, and GT = 1.000000; gamma distribution shape parameter α = 0.294728. Bayesian analysis resulted in the average standard deviation of split frequencies as 0.009934.

Analysis 2: The combination of ITS, LSU, and TEF1-α sequence dataset comprised 156 taxa of Apiospora, and other related taxa in Apiosporaceae. Sporocadus trimorphus (CBS 114203) were selected as the outgroup taxon (Figure S1). The final alignment consisted of 2422 total characters, including gaps (ITS: 1–641 bp, LSU: 642–1524 bp, TEF1-α: 1525–2422 bp). The RAxML analysis of the integrated dataset yielded a best scoring tree with a final ML optimization likelihood value of −24,247.334486. The aligned sequence matrix contained 1182 distinct alignment patterns, with 37.22% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.245462, C = 0.240858, G = 0.258736, T = 0.254944; substitution rates AC = 1.136736, AG = 2.122936, AT = 1.134884, CG = 1.008342, CT = 4.360445, and GT = 1.000000; gamma distribution shape parameter α = 0.245115. Bayesian analysis resulted in the average standard deviation of split frequencies as 0.009872.

Analysis 3: The combination of ITS, LSU, TUB2, and TEF1-α sequence dataset comprised 31 taxa in Apiospora locuta-pollinis/marii clade. Apiospora fermenti KUC 2189 and A. pseudospegazzinii CBS 102052 were selected as the outgroup taxa (Figure 2). The final alignment consisted of 2735 total characters, including gaps (ITS: 1–626 bp, LSU: 627–1470 bp, TUB2: 1471–2296 bp, TEF1-α: 2297–2735 bp). The RAxML analysis of the integrated dataset yielded a best scoring tree with a final ML optimization likelihood value of −5544.668718. The aligned sequence matrix contained 363 distinct alignment patterns, with 25.58% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.237367, C = 0.256117, G = 0.247967, T = 0.258550; substitution rates AC = 1.390352, AG = 3.604609, AT = 1.633776, CG = 0.564937, CT = 4.605733, and GT = 1.000000; gamma distribution shape parameter α = 0.020000. The maximum parsimonious dataset consisted of 2744 characters of which 2523 were constant, 119 parsimony-informative and 102 parsimony-uninformative. The parsimony analysis of the data matrix resulted in a single most parsimonious tree (TL = 266, CI = 0.883, RI = 0.914, RC = 0.808, HI = 0.117). Bayesian analysis resulted in the average standard deviation of split frequencies as 0.009680.

Phylogenetic analyses inferred from ML and BI analyses were not significantly different and showed congruent topologies. The overall tree topologies of the concatenated ITS-LSU-TUB2-TEF1-α sequence dataset (Figure 1) were also congruent with the tree topologies of a concatenated ITS-LSU-TEF1-α sequence matrix (Figure S1). However, the first analysis (Figure 1) revealed higher statistical support than that from the second analysis (Figure S1). Therefore, the phylogenetic results of the concatenated ITS-LSU-TUB2-TEF1-α sequence matrix was selected for discussion in this study. Phylogenetic results showed that the new species, Apiospora mukdahanensis formed a well-resolved clade sister to A. mori with significant support (100% ML/1.00 PP, Figure 1). The new strains, KUNCC 22-12408 and KUNCC 22-12409 clustered in the same clade with A. locuta-pollinis including the ex-type strain (LC 11683) with 85% ML support (Figure 1).

The multigene phylogeny based on the ITS-LSU-TUB2-TEF1-α sequence data of the species in the first clade (Apiospora locuta-pollinis/marii clade) revealed a similar result between ML, MP, and BI analyses. The results indicated that A. locuta-pollinis formed a well-supported clade with 75% ML/77% MP/0.98 PP support including the new strain KUNCC 22-12408 which formed a separated branch basal to other A. locuta-pollinis strains and the strain KUNCC 22-12409 shared the same branch length with A. locuta-pollinis (LC 11688) (Figure 2), whereas the type of A. hispanica, A. mediterranea, and A. marii were not well separated and clustered together with low support (Figure 2). Apiospora gaoyouensis formed an independent clade basal to A. marii with significant support (100% ML/100% MP/1.00 PP, Figure 2). The two strains of A. marii (CBS 113535 and CBS 114803) grouped within A. locuta-pollinis clade (Figure 2).

3.2. Taxonomy

3.2.1. Apiospora mori (Tennakoon, C.H. Kuo and K.D. Hyde) Monkai and Phookamsak, comb. nov.

Index Fungorum number: IF559913; Facesoffungi number: FoF 12871

Basionym: Arthrinium mori Tennakoon, C.H. Kuo and K.D. Hyde, Fungal diversity 108: 215 (2021).

Notes: Arthrinium mori was introduced by Tennakoon et al. [60] from dead leaves of Morus australis in Taiwan. Tennakoon et al. [60] noted that Ar. mori forms a well-supported branch sister to Ar. jatrophae with 86% ML/1.00 PP support, but differs from Ar. jatrophae in having smaller conidia (4.5–5.5 × 4–5 vs. 6.5–9.7 × 3.2–6.5 μm) [6]. In our phylogenetic analyses, Ar. mori constitutes an independent clade sister to Apiospora mukdahanensis with high support (100% ML/1.00 PP, Figure 1). Based on the phylogenetic analysis, 55 Arthrinium species were proposed as new combinations of Apiospora, but they did not include Ar. mori [2]. Thus, we transferred Ar. mori under the new combination A. mori, on the basis of morphological and molecular data.

3.2.2. Apiospora mukdahanensis Monkai and Phookamsak, sp. nov.

Index Fungorum number: IF559912; Facesoffungi number: FoF 12853; Figure 3.

Etymology: Referring to the locality, Mukdahan Province, Thailand, where the holotype was collected.

Holotype: MFLU 22-0104

Saprobic on dead bamboo leaves. Sexual morph: Undetermined. Asexual morph: Conidiomata sporodochia, rounded to ovoid, pulvinate, dark brown to black, 150–400 μm diam. Mycelium basal, consists of smooth, brown to dark brown, branched septate hyphae. Conidiophore mother cells arising from the mycelial mat, obpyriform to lageniform, dark brown, smooth, (6–)7–8.5(–10.5) × (2.5–)3.5–4(–5.5) μm (x = 8.5 × 4 μm, n = 12). Conidiophores basauxic, arising from conidiophore mother cells, cylindrical, pale brown, septate, with brown transversal septa, straight or flexuous. Conidiogenous cells polyblastic, smooth, hyaline to pale brown, ampulliform, cylindrical or lageniform, (6–)8.5–14(–20) × 1.5–2(–3) μm (x = 11 × 2 μm, n = 15). Conidia brown to dark brown, smooth to slightly roughened, thick-walled, globose to subglobose, or irregularly round in face view, lenticular in side view (6–)–7.5(–8) × (5–)–6.5(–7) μm (x = 7 × 6 μm, n = 30), with a pale equatorial slit, and a central scar.

Culture characteristics: Colonies on PDA reached at 9 cm diam. in 2 weeks at 28 °C, flat, fluffy, spreading, with abundant aerial mycelium, irregular margin, white to cream, in reverse, white and pale yellowish in the center.

Material examined: THAILAND, Mukdahan Province, Tambon Phang Daeng, Amphoe Dong Luang, on dead bamboo leaves, 24 July 2019, E. Yasanthika, E2B-4 (MFLU 22-0104, holotype), ex-type living culture, MFLUCC 22-0056.

Notes: The nucleotide BLAST search of ITS region showed that Apiospora mukdahanensis (MFLUCC 22-0056) has high similarity with Arthrinium sp. strain 4–13 (99.19%), Arthrinium sp. strain NF34_TK10 and A. mori strain MFLU 18-2514 (98.47%). The nucleotide BLAST search of LSU region showed that A. mukdahanensis (MFLUCC 22-0056) has high similarity with Apiospora sp. strain NF34_TK10 (99.03%), Apiospora sp. strain JYZ-2021a (98.95%) and A. mori MFLU 18-2514 (98.83%). The nucleotide BLAST search of TEF1-α region showed that A. mukdahanensis (MFLUCC 22-0056) has high similarity with A. phragmitis strain MFLUCC 18-0099 (95.96%), A. locuta-pollinis strain LC 11689, LC 11688 (95.92%), and Arthrinium sp. strain MFLU 18-2333 (95.64%).

Based on phylogenetic analysis, Apiospora mukdahanensis formed an independent lineage sister to A. mori with 100% ML and 1.00 PP support (Figure 1). Morphologically, Apiospora mukdahanensis is distinct from A. mori in having larger conidia (6–8.1 × 5.1–6.9 vs. 4.5–5.5 × 4–5 μm) with slightly roughened wall, whereas A. mori has smooth-walled conidia [60]. The new species resembles A. jatrophae in having overlapping size range of conidia (6–8.1 × 5.1–6.9 vs. 6.5–9.7 × 3.2–6.5 μm) [6]. However, A. jatrophae differs from A. mukdahanensis in having thick multi-septate conidiophores with brown transverse septa and two types of conidia including smooth-walled, lenticular conidia and anomalous conidia [6]. The base-pair comparison of ITS gene region showed 4.1% base pair differences (21/508 bp) between A. mukdahanensis and A. mori (MFLUCC 20-0181 and NCYUCC 19-0340) and 9.6% base pair differences (48/498 bp) between A. mukdahanensis and A. jatrophae (AMH-9556, AMH-9557). However, the TEF1-α sequence data are not available for A. mori and A. jatrophae to compare with our new species.

3.2.3. Apiospora locuta-pollinis (F. Liu and L. Cai) Pintos and P. Alvarado, Fungal Systematics and Evolution 7: 206 (2021)

Index Fungorum number: IF837763; Facesoffungi number: FoF 05221, Figure 4.

Basionym: Arthrinium locuta-pollinis F. Liu and L. Cai, Mycosphere 9(6): 1094 (2018).

Saprobic on decaying stems of bamboo. Sexual morph: Immature state found associated with asexual morph on host. Asexual morph: Conidiomata associated with the sexual morph, pycnidial, raised, stromatic at base, 150–230 µm high, 450–830 µm diam., covered by black conidial masses on host surface, laying parallel to the longitudinal axis of the stem, clustered, solitary to gregarious, subepidermal to erumpent, globose to subconical, or hemispherical, uni- to multi-loculate; individual pycnidium 200–260 µm diam., 150–210 µm high, glabrous, ostiolate, opening by longitudinal splitting of the epidermis. Pycnidial wall (15–)20–40(–50) µm wide, of unequal thickness, slightly thick at sides towards apex, thinner at base, composed of several cell layers of reddish-brown to dark brown pseudoparenchymatous cells of textura angularis to textura prismatica, with outer layers intermixed with the host’s tissues. Conidiophore mother cells (3.5–)4.5–9(–12) × 3.5–5.5 µm (x = 7.8 × 4.8 µm, n = 30), arising in dense packs from hyaline to light brown, irregularly angled palisade-like cells (4–9 × (2.8–)3–4.5 µm) in the stroma, subhyaline to pale brown, ampulliform to cylindrical, tapering towards rounded apex with small granules. Conidiophores (4–)5–10(–20) × (1.5–)2–3.5 µm (x = 8.9 × 2.2 µm, n = 30), basauxic, arising from conidiophore mother cells, conspicuous, short, septate, branched, smooth, pale brown, flexuous. Conidiogenous cells (3.5–)4.5–6.5(–9) × 2.5–4.5 µm (x = 6 × 3.5 µm, n = 30), polyblastic, straight or flexuous, cylindrical to subcylindrical or ampulliform, hyaline to light brown, smooth or with small granules, moderately brown, denticulate, sometimes flattened. Conidia (8–)9–13 × (7–)9–12 µm (x = 10.5 × 10 µm, n = 50), globose to obovoid, or ellipsoidal, dark brown, smooth-walled, with a paler equatorial slit, sometimes small piece of the denticle remains attached to the base of the conidium, (1–)2–5(–10) × 1.5–3 µm ($\overline{x}$ = 3.8 × 1.9 µm, n = 20). Sterile cells are brown, leaf-like, attached to the conidiophore.

Material examined: CHINA, Yunnan Province, Honghe Autonomous Prefecture, Honghe County, Honghe Hani Rice Terraces, on decaying stem of bamboo, 26 January 2021, R. Phookamsak, bn01 (KUN-HKAS 124566), living culture = KUNCC 22-12408; Honghe Hani Rice Terraces, on dead stem of bamboo, 26 January 2021, R. Phookamsak, bn11 (KUN-HKAS 124567), living culture = KUNCC 22-12409.

Notes: The nucleotide BLAST search of ITS region showed that Apiospora locuta-pollinis (KUNCC 22-12408 and KUNCC 22-12409) has high similarity with A. marii strain CBS 497.90, isolate A4, CPC 18904, CPC 18902, CBS 200.57, CBS 113535, Arthrinium sp. strain MFLUCC 16-0282, Fungal sp. BMP3011 (100%). The nucleotide BLAST search of LSU region showed that A. locuta-pollinis (KUNCC 22-12408 and KUNCC 22-12409) has high similarity with A. guizhouensis strain LC5318, A. locuta-pollinis isolate SICAUCC 22-0037, A. sacchari strain CBS 664.74, CBS 372.67, CBS 301.49, CBS 212.30, A. marii strain CBS 113535, A. biserialis isolate CS19-17 and A. guizhouensis strain KUMCC 20-0206 (100%). The nucleotide BLAST search of TEF1-α region showed that A. locuta-pollinis (KUNCC 22-12408) has high similarity with A. marii isolate GUCC 10214 (100%) and A. locuta-pollinis (KUNCC 22-12409) has high similarity with A. locuta-pollinis strain LC 11683, A. marii culture-collection CBS 113535, CBS 114803, GUCC 10254, GUCC 10227, A. locuta-pollinis strain LC 11688, LC 11689 (100%).

In our multigene phylogeny, two new strains (KUNCC 22-12408 and KUNCC 22-12409) shared a close relationship with Apiospora locuta-pollinis based on ML, MP, and BI analyses (Figure 1 and Figure 2). The base-pair comparison of ITS gene regions indicated that strain KUNCC 22-12408 is identical to strain KUNCC 22-12409 and other A. locuta-pollinis strains, except for a 1 bp difference with GUCC 10228. In the base-pair comparison of TEF1-α gene regions, strain KUNCC 22-12409 had no base pair differences with all the A. locuta-pollinis strains and strain KUNCC 22-12408 had a 2 bp difference (0.5%) with KUNCC 22-12409 and other strains of A. locuta-pollinis.

Morphological characteristics of our new strains (KUNCC 22-12408 and KUNCC 22-12409) were compared with the type strain of Apiospora locuta-pollinis (LC 11683) (Table 2). Both new strains are similar to the type strain of A. locuta-pollinis in having globose to subglobose conidia with hyaline equatorial rim, however they have larger conidia (10.5 × 10 and 9.6 × 8 vs. 7.1 × 6.4 μm) (Table 2). The conidiogenous cells of strain KUNCC 22-12409 are more similar to the type strain (LC 11683) in being subglobose to ampulliform to doliiform, but with smaller size (3.2 × 2.2 vs. 4.9 × 3.8 μm) (Table 2)., whereas the strain KUNCC 22-12408 has cylindrical to subcylindrical or ampulliform conidiogenous cells with larger size compared to other strains (6 × 3.5 µm) (Table 2). The morphological description of A. locuta-pollinis was based on cultures and its conidiophores were reduced to conidiogenous cells [61]. Thus, the comparisons of conidiomata and conidiophores characteristics between these strains were not possible. In addition, we found some morphological differences between two new strains including strain KUNCC 22-12408 which had longer conidiophore mother cells and conidiogenous cells, but shorter conidiophores compared to strain KUNCC 22-12409 (Table 2).

Apiospora locuta-pollinis was previously isolated from hive-stored pollen of Brassica campestris in Hubei province, China [61], whereas the new strains KUNCC 22-12408 were isolated from decaying bamboo and KUNCC 22-12409 was isolated from dead bamboo in Yunnan province, China. Although there were some morphological variations among the new strains and the type strain of A. locuta-pollinis, the multigene phylogeny and DNA sequence comparisons (ITS and TEF1-α gene regions) did not provide the necessary support to delineate them as a distinct species. Therefore, we treated these strains as new records of A. locuta-pollinis. It is possible that the strain KUNCC 22-12408 is a new species due to the significant morphological and phylogenetic differences, which caused it to form a separated branch to other A. locuta-pollinis strains. Further taxonomic studies are needed to resolve their conspecific status.

3.3. Host and Geographical Distribution of Apiospora Species

Based on species distribution data, Apiospora is widely distributed in temperate, subtropical, and tropical areas, including Africa, America, Asia, Australia, and Europe (Figure 5). The highest species diversity of Apiospora was found in Asia (e.g., China, India, Japan, Thailand) (Figure 5). However, the data reflect areas in which there have been reports of Apiospora species and may thus reflect hotspots of research, and not just species hotspots. Areas shown to be devoid of Apiospora species may just be areas that require further study. The host-substratum diversity of Apiospora species (Figure 6) showed that most species have been found exclusively associated with Poaceae (63%), including bamboo (31%) and non-bamboo (32%). The most common bamboo genera associated with Apiospora are Bambusa, Phyllostachys, and Arundinaria and the most common non-bamboo genera are Saccharum, Phragmites, and Arundo.

4. Discussion

Our study provides better insight into interspecific and intraspecific variation in Apiospora, particularly in the A. locuta-pollinis/marii clade. Phylogenetic analyses of four gene markers (ITS, LSU, TUB2, and TEF1-α) revealed the distinct relationships between A. marii, A. locuta-pollinis, and A. gaoyouensis (Figure 1 and Figure 2). Morphologically, they have similar conidia characteristics (globose to elongate ellipsoid in surface, lenticular in side view) with an overlapping size range: 7.2–7.5 µm diam. in Apiospora marii, 5.5–9 µm diam. in A. locuta-pollinis, and 5–8 µm diam. in A. gaoyouensis (Table 3).

In the Apiospora marii clade, A. hispanica and A. mediterranea are not well-resolved (Figure 1 and Figure 2). Morphologically, A. marii and A. hispanica have overlapping sizes of conidia (7.2–7.5 × 6.1–6.5 vs. 7.5–8.5 × 6.2–7.6 µm) (Table 3), whereas A. mediterranea has larger conidia (9–9.5 × 7.5–9 µm) (Table 3). The base-pair difference of ITS and TUB2 sequence data indicated that they are consistent. However, the LSU sequence data of A. hispanica and A. mediterranea are in short length (320 base pairs) and their TEF1-α sequence data were lacking. The morphological reexamination and molecular data of the type specimens of A. hispanica and A. mediterranea are required to confirm a putative synonymy.

In the Apiospora locuta-pollinis clade, two strains of A. marii CBS 113535 and CBS 114803 clustered together (Figure 2). Pintos et al. [5] and Garin et al. [62] also reported that A. marii CBS 114803 had a well-supported lineage distant from the A. marii clade. Tian et al. [12] synonymized A. pseudomarii GUCC 10228 as A. locuta-pollinis based on morphology and phylogeny. Crous and Groenewald [3] provided the sequence data of these strains in which CBS 113535 was isolated from oats in Sweden and CBS 114803 was isolated from the culm of Arundinaria hindsi in Hongkong. Thus, we treated these strains as A. locuta-pollinis based on the phylogenetic evidence.

TUB2 and TEF1-α gene regions are essential phylogenetic markers for accurate identification of Apiospora species [3,4,5,9,38]. Most of the recent studies have used multigene phylogenetic analyses of integrated ITS, LSU, TUB2, and TEF1-α sequence data for Apiospora species delineation [2,5,10,11,12,24,25,38]. However, our phylogenetic analysis based on the integrated ITS, LSU, and TEF1-α sequence data also provided the necessary resolution to distinguish species of Apiospora (Figure S1). In addition, the TEF1-α gene region could support the species delineation between A. marii and A. locuta-pollinis. As they have a 10 bp difference (2.3%) in the TEF1-α gene region, whereas no difference was found in TUB2 gene region. It seems that the TUB2 gene region is uninformative for the separation of species in the A. locuta-pollinis/marii clade. Nevertheless, with the lack of TUB2 sequences in our new strains, the TEF1-α gene region was not enough to resolve their placements within A. locuta-pollinis lineage. We suggest that TUB2 gene and other protein-coding genes, such as RPB2, should be included for further phylogenetic analysis to confirm their actual identity and placements.

Our study also revealed significant morphological variation among Apiospora locuta-pollinis strains. This result was also observed for other Apiospora species. For instance, A. yunnana, introduced by Dai et al. [8], based on a sexual morph on bamboo culms, had larger conidia in cultures (15.5–26.5 μm diam.), compared to the strains CFCC 52311, CFCC 5231 which were isolated and described directly from bamboo culms (10–16 μm diam.) [9]. Apiospora pseudoparenchymatica, introduced by Wang et al. [4], and was isolated from living leaves of bamboo and described by its asexual morph. Feng et al. [11] found a new record of A. pseudoparenchymatica from decaying bamboo culms and noted the significant difference in the characteristics of conidiophores and conidiogenous cells of the new strain, GZCC 20–0117 (on WA) compared to the type specimen, LC 8173 (on PDA and MEA) [4]. The significant variation in morphology might be due to the differences in substrates (natural substrates or cultures), growth conditions, hosts, and habitats. This observation makes our finding all the more valuable, we found both new strains from the same host substrate and habitat. The only difference is strain KUNCC 22-12408, which was from the decaying state and strain KUNCC 22-12409 was from the dead state of bamboo. Therefore, our study highlighted the great impact of environmental factors on morphological variation.

Geographically, Asia was found to be home to greatest diversity of Apiospora (Figure 5) and is likely the result of the rich diversity of bamboo genera/species, especially those in China [63]. More extensive sampling of these hosts will surely result in the discovery of additional new species. Although the host preference of Apiospora is the family Poaceae, there remains a number of species which have been found on other plant families (Figure 6), such as A. euphorbiae (on Euphorbiaceae, Lauraceae, Pinaceae, Zingiberaceae) [64], and A. jiangxiensis (on Lauraceae, Primulaceae, Theaceae) [4]., On the contrary, some species seem to be host-specific, such as A. pterosperma which has only been found on Cyperaceae (Lepidosperma and Machaerina) [3], and A. rhododendri has only been reported from Ericaceae (Rhododendron) [64]. Some species are ubiquitous, with a diverse range of hosts and habitats (e.g., A. arundinis, A. marii, A. rasikravindrae, A. serenensis) [2,3,4,5,8,12,64]. It is noteworthy that the asexual morph is more frequently discovered from different ecological habitats, and it is possible they are the cause of certain plant diseases [2,62]. For example, A. arundinis which has been isolated from soil, water, and numerous plant hosts [4,64], is also reported as causing some plant diseases, such as brown culm streak of Phyllostachys praecox [65] and the leaf spot of rosemary (Salvia rosmarinus) [66]. Apiospora marii has been isolated from air, sand, and different plant hosts [3,5], also reported as causing die-back of olives (Olea europaea) [62].

5. Conclusions

Based on the rate of discovery, their diverse host ranges and different life-styles, the species number of Apiospora is likely to continue to increase in the future [39,48]. There are still a number of regions that have remained unstudied in terms of Apiospora, which will likely further add to the current list of species within this genus. A comprehensive survey of these unknown regions along with a polyphasic taxonomic study of Apiospora is necessary, especially focusing on Poaceae species, will enable a better understanding of host relationships and the ecological significance of this group of fungi.

Author Contributions

Conceptualization, J.M. and R.P.; data curation, J.M.; formal analysis, J.M., D.S.T. and S.X.; funding acquisition, J.X. and S.L.; investigation, J.M., R.P., S.X. and Q.L.; methodology, J.M. and R.P.; project administration, J.M. and R.P.; resources, J.M., R.P., S.X. and Q.L.; supervision, J.X., J.K. and S.L.; validation, J.M., R.P., D.J.B. and S.L.; writing—original draft, J.M. and R.P.; writing—review and editing, J.M., R.P., D.S.T., D.J.B., P.E.M. and J.K. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data can be found within the manuscript.

Acknowledgments

J.M. would like to thank the Post–Doctoral Fellowship 2022 from Chiang Mai University, Thailand (Grant No. R000030592). R.P. would like to thank the Post–Doctoral Fellowship 2022 from Chiang Mai University, Thailand (Grant No. R000031743). J.M. is grateful to Erandi Yasanthika and Areerat Manowong for their assistance during this research. Shaun Pennycook from Landcare Research, Auckland, New Zealand, is thanked for advising on the taxon name. We also acknowledge the Biology Experimental Center, Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences for providing the molecular laboratory facilities.

Conflicts of Interest

The authors declare no conflict of interest.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures and Tables

Figure 1. Phylogenetic tree retrieved from RAxML analyses of a combined ITS, LSU, TUB2, and TEF1-α data sequence of Apiospora, and other related taxa in the family Apiosporaceae. Bootstrap support values for ML equal or greater than 60% and Bayesian posterior probabilities greater than 0.90 are indicated at the nodes as ML/PP. The ex-type strains are in bold. The new species are in red and new record and new combination species are in blue. The tree is rooted to Sporocadus trimorphus (CBS 114203).

Figure 1. Phylogenetic tree retrieved from RAxML analyses of a combined ITS, LSU, TUB2, and TEF1-α data sequence of Apiospora, and other related taxa in the family Apiosporaceae. Bootstrap support values for ML equal or greater than 60% and Bayesian posterior probabilities greater than 0.90 are indicated at the nodes as ML/PP. The ex-type strains are in bold. The new species are in red and new record and new combination species are in blue. The tree is rooted to Sporocadus trimorphus (CBS 114203).

Figure 2. Phylogenetic tree retrieved from RAxML analyses of a combined ITS, LSU, TUB2, and TEF1-α data sequence of taxa in Apiospora locuta-pollinis/marii clade. Bootstrap support values for ML and MP equal or greater than 60% and Bayesian posterior probabilities greater than 0.90 are indicated at the nodes as ML/MP/PP. The ex-type strains are in bold. The new record strains are in blue. The tree is rooted to Apiospora fermenti KUC 2189 and A. pseudospegazzinii CBS 102052.

Figure 3. Apiospora mukdahanensis (MFLU 22-0104, holotype). (a) Conidiomata on host substrate. (b) Conidiophore mother cells. (c–e) Conidiophores, conidiogenous cells and conidia. (f–k) Conidia. (l,m) Colonies on PDA ((l) from above, (m) from reverse). Scale bars: (a) = 200 μm, (b–k) = 5 μm.

Figure 4. Apiospora locuta-pollinis (KUN-HKAS 124566). (a) Conidiomata on host substrate. (b,c) Section through the stromata. (d) Section through pycnidial wall. (e) Conidiophore mother cells. (f,g) Conidiogenous cells giving rise conidia. (h–j) Conidiophores (sterile cells attached to the conidiophore in (i,j)). (k,l) Conidia (equatorial slit is indicated by arrows in (k)). Scale bars: (b,c) = 100 µm, (d) = 50 µm, (e–l) = 10 µm.

Figure 5. Species diversity hotspot countries of Apiospora.

Figure 6. Host-substratum diversity of Apiospora species.

Table 1

List of the taxa used in phylogenetic reconstruction and their corresponding GenBank numbers.

Taxa	Culture AccessionNo.	Genbank Accession No.
		ITS	LSU	TUB2	TEF1-α
Apiospora acutiapica	KUMCC 20-0210	MT946343	MT946339	MT947366	MT947360
Apiospora agari	KUC 21333	MH498520	N/A	MH498478	MH544663
Apiospora aquatica	S-642	MK828608	MK835806	N/A	N/A
Apiospora arctoscopi	KUC 21331	MH498529	N/A	MH498487	MN868918
Apiospora arundinis	CBS 133509	KF144886	KF144930	KF144976	KF145018
Apiospora arundinis	CBS 449.92	KF144887	KF144931	KF144977	KF145019
Apiospora aurea	CBS 244.83	AB220251	KF144935	KF144981	KF145023
Apiospora balearica	CBS 145129	MK014869	MK014836	MK017975	MK017946
Apiospora bambusae	CBS 145133	MK014875	MK014842	MK017981	MK017952
Apiospora bambusae	ICMP 6889	MK014874	MK014841	MK017980	MK017951
Apiospora bambusicola	MFLUCC 20-0144	MW173030	MW173087	N/A	MW183262
Apiospora biserialis	CGMCC 3.20135 *	MW481708	MW478885	MW522955	MW522938
Apiospora biserialis	GZCC 20_0099 *	MW481709	MW478886	MW522956	MW522939
Apiospora camelliae-sinensis	LC 5007	KY494704	KY494780	KY705173	KY705103
Apiospora camelliae-sinensis	LC 8181	KY494761	KY494837	KY705229	KY705157
Apiospora chiangraiense	MFLUCC 21-0053	MZ542520	MZ542524	MZ546409	N/A
Apiospora chromolaenae	MFLUCC 17-1505	MT214342	MT214436	N/A	MT235802
Apiospora cordylinae	GUCC 10026	MT040105	N/A	MT040147	MT040126
Apiospora cyclobalanopsidis	CGMCC 3.20136	MW481713	MW478892	MW522962	MW522945
Apiospora descalsii	CBS 145130	MK014870	MK014837	MK017976	MK017947
Apiospora dichotomanthi	LC 4950	KY494697	KY494773	KY705167	KY705096
Apiospora dichotomanthi	LC 8175	KY494755	KY494831	KY705223	KY705151
Apiospora esporlensis	CBS 145136	MK014878	MK014845	MK017983	MK017954
Apiospora euphorbiae	IMI 285638b	AB220241	AB220335	AB220288	N/A
Apiospora fermenti	KUC 21289 *	MF615226	N/A	MF615231	MH544667
Apiospora gaoyouensis	CFCC 52301 *	MH197124	N/A	MH236789	MH236793
Apiospora gaoyouensis	CFCC 52302 *	MH197125	N/A	MH236790	MH236794
Apiospora garethjonesii	KUMCC 16-0202	KY356086	KY356091	N/A	N/A
Apiospora gelatinosa	KHAS 11962 *	MW481706	MW478888	MW522958	MW522941
Apiospora guiyangensis	HKAS 102403	MW240647	MW240577	MW775604	MW759535
Apiospora guizhouensis	LC 5318 *	KY494708	KY494784	KY705177	KY705107
Apiospora guizhouensis	LC 5322 *	KY494709	KY494785	KY705178	KY705108
Apiospora hispanica	IMI 326877 *	AB220242	AB220336	AB220289	N/A
Apiospora hydei	CBS 114990	KF144890	KF144936	KF144982	KF145024
Apiospora hydei	LC 7103	KY494715	KY494791	KY705183	KY705114
Apiospora hyphopodii	MFLUCC 15-0003	KR069110	N/A	N/A	N/A
Apiospora hyphopodii	KUMCC 16-0201	KY356088	KY356093	N/A	N/A
Apiospora ibericum	CBS 145137	MK014879	MK014846	MK017984	MK017955
Apiospora intestini	CBS 135835	KR011352	KR149063	KR011350	KR011351
Apiospora intestini	MFLUCC 21-0052	MZ542521	MZ542525	MZ546410	MZ546406
Apiospora italica	CBS 145138	MK014880	MK014847	MK017985	MK017956
Apiospora italica	CBS 145139	MK014881	MK014848	MK017986	MK017957
Apiospora jatrophae	AMH-9556	HE981191	N/A	N/A	N/A
Apiospora jatrophae	AMH-9557	JQ246355	N/A	N/A	N/A
Apiospora jiangxiense	LC 4494	KY494690	KY494766	KY705160	KY705089
Apiospora jiangxiense	LC 4577	KY494693	KY494769	KY705163	KY705092
Apiospora kogelbergense	CBS 113332	KF144891	KF144937	KF144983	KF145025
Apiospora kogelbergense	CBS 113333	KF144892	KF144938	KF144984	KF145026
Apiospora koreana	KUC 21332	MH498524	N/A	MH498482	MH544664
Apiospora locuta-pollinis	GUCC 10228 *	MT040124	N/A	MT040166	MT040145
Apiospora locuta-pollinis	KUNCC 22-12408 *	OP377736	OP377743	N/A	OP381090
Apiospora locuta-pollinis	KUNCC 22-12409 *	OP377737	OP377744	N/A	OP381091
Apiospora locuta-pollinis	LC 11683 *	MF939595	N/A	MF939622	MF939616
Apiospora locuta-pollinis	LC 11688 *	MF939596	N/A	MF939623	MF939618
Apiospora longistromum	MFLUCC 11-0479 *	KU940142	KU863130	N/A	N/A
Apiospora longistromum	MFLUCC 11-0481 *	KU940141	KU863129	N/A	N/A
Apiospora malaysiana	CBS 102053	KF144896	KF144942	KF144988	KF145030
Apiospora marii	CBS 113535 *	KF144898	KF144944	KF144990	KF145032
Apiospora marii	CBS 114803 *	KF144899	KF144945	KF144991	KF145033
Apiospora marii	CPC 18902 *	KF144901	KF144948
Apiospora marii	CPC 18904 *	KF144902	KF144949	KF144994	KF145036
Apiospora marii	CBS 200.57 *	KF144900	KF144946	KF144992	KF145034
Apiospora marii	CBS 497.90 *	AB220252	KF144947	KF144993	KF145035
Apiospora marii	KUC 21338 *	MH498549	N/A	MH498507	MH544681
Apiospora marii	MFLUCC 16-0282 *	MH109526	N/A	N/A	MH206166
Apiospora marii	MFLUCC 16-0283 *	MH109527	N/A	N/A	MH220419
Apiospora marina	KUC 21328	MH498538	N/A	MH498496	MH544669
Apiospora mediterranea	IMI 326875 *	AB220243	AB220337	AB220290	N/A
Apiospora minutispora	17E-042	LC517882	N/A	LC518888	LC518889
Apiospora mori	NCYUCC 19-0340	MW114314	MW114394	N/A	N/A
Apiospora mori	MFLUCC 20-0181	MW114313	MW114393	N/A	N/A
Apiospora mukdahanensis	MFLUCC 22-0056	OP377735	OP377742	N/A	OP381089
Apiospora multiloculata	MFLUCC 21-0023	OL873137	OL873138	OL874718	N/A
Apiospora mytilomorpha	DAOM 214595	KY494685	N/A	N/A	N/A
Apiospora neobambusae	LC 7106	KY494718	KY494794	KY705186	KY806204
Apiospora neobambusae	LC 7124	KY494727	KY494803	KY705195	KY806206
Apiospora neochinensis	CFCC 53036	MK819291	N/A	MK818547	MK818545
Apiospora neochinensis	CFCC 53037	MK819292	N/A	MK818548	MK818546
Apiospora neogarethjonesii	KUMCC 18-0192	MK070897	MK070898	N/A	N/A
Apiospora neosubglobosa	JHB006	KY356089	KY356094	N/A	N/A
Apiospora neosubglobosa	KUMCC 16-0203	KY356090	KY356095	N/A	N/A
Apiospora obovata	LC 4940	KY494696	KY494772	KY705166	KY705095
Apiospora obovata	LC 8177	KY494757	KY494833	KY705225	KY705153
Apiospora ovata	CBS 115042	KF144903	KF144950	KF144995	KF145037
Apiospora paraphaeospermum	MFLUCC 13-0644	KX822128	KX822124	N/A	N/A
Apiospora phragmitis	CPC 18900	KF144909	KF144956	KF145001	KF145043
Apiospora phyllostachydis	MFLUCC 18-1101	MK351842	MH368077	MK291949	MK340918
Apiospora piptatheri	CBS 145149 *	MK014893	MK014860	N/A	MK017969
Apiospora pseudoparenchymatica	LC 7234	KY494743	KY494819	KY705211	KY705139
Apiospora pseudoparenchymatica	LC 8173	KY494753	KY494829	KY705221	KY705149
Apiospora pseudorasikravindrae	KUMCC 20-0208	MT946344	N/A	MT947367	MT947361
Apiospora pseudosinensis	CPC 21546	KF144910	KF144957	N/A	KF145044
Apiospora pseudospegazzinii	CBS 102052 *	KF144911	KF144958	KF145002	KF145045
Apiospora pterosperma	CBS 123185	KF144912	KF144959	KF145003	N/A
Apiospora pterosperma	CPC 20193	KF144913	KF144960	KF145004	KF145046
Apiospora pusillisperma	KUC 21321	MH498533	N/A	MH498491	MN868930
Apiospora qinlingense	CFCC 52303	MH197120	N/A	MH236791	MH236795
Apiospora qinlingense	CFCC 52304	MH197121	N/A	MH236792	MH236796
Apiospora rasikravindrae	NFCCI 2144	JF326454	N/A	N/A	N/A
Apiospora rasikravindrae	LC 8179	KY494759	KY494835	KY705227	KY705155
Apiospora sacchari	CBS 372.67 *	KF144918	KF144964	KF145007	KF145049
Apiospora sacchari	CBS 664.74 *	KF144919	KF144965	KF145008	KF145050
Apiospora saccharicola	CBS 191.73	KF144920	KF144966	KF145009	KF145051
Apiospora saccharicola	CBS 831.71	KF144922	KF144969	KF145012	KF145054
Apiospora sargassi	KUC 21228	KT207746	N/A	KT207644	MH544677
Apiospora septata	CGMCC 3.20134 *	MW481711	MW478890	MW522960	MW522943
Apiospora serenensis	IMI 326869	AB220250	AB220344	AB220297	N/A
Apiospora setariae	CFCC 54041	MT492004	N/A	MT497466	MW118456
Apiospora setostroma	KUMCC 19-217	MN528012	MN528011	N/A	MN527357
Apiospora sichuanensis	HKAS 107008	MW240648	MW240578	MW775605	MW759536
Apiospora sorghi	URM 93000	MK371706	N/A	MK348526	N/A
Apiospora subglobosa	MFLUCC 11-0397	KR069112	KR069113	N/A	N/A
Apiospora subrosea	LC 7291	KY494751	KY494827	KY705219	KY705147
Apiospora subrosea	LC 7292	KY494752	KY494828	KY705220	KY705148
Apiospora taeanense	KUC 21322	MH498515	N/A	MH498473	MH544662
Apiospora thailandica	MFLUCC 15-1999	KU940146	KU863134	N/A	N/A
Apiospora thailandica	MFLUCC 15-0202	KU940145	KU863133	N/A	N/A
Apiospora tropica	MFLUCC 21–0056	OK491657	OK491653	OK560922	N/A
Apiospora vietnamense	IMI 99670	KX986096	KX986111	KY019466	N/A
Apiospora xenocordella	CBS 478.86	KF144925	KF144970	KF145013	KF145055
Apiospora xenocordella	CBS 595.66	KF144926	KF144971	N/A	N/A
Apiospora yunnana	MFLUCC 15-1002	KU940147	KU863135	N/A	N/A
Apiospora yunnana	DDQ00281	KU940148	KU863136	N/A	N/A
Arthrinium austriacum	GZU 345004	MW208928	N/A	N/A	N/A
Arthrinium austriacum	GZU 345006	MW208929	MW208860	N/A	N/A
Arthrinium caricicola	CBS 145127	MK014871	MK014838	MK017977	MK017948
Arthrinium cf. sporophleoides	GZU 345102	MW208944	MW208866	N/A	N/A
Arthrinium crenatum	CBS 146353	MW208931	MW208861	MW221923	MW221917
Arthrinium curvatum	AP 25418	MK014872	MK014839	MK017978	MK017949
Arthrinium japonicum	IFO 30500	AB220262	AB220356	AB220309	N/A
Arthrinium japonicum	IFO 31098	AB220264	AB220358	AB220311	N/A
Arthrinium luzulae	AP 7619-3	MW208937	MW208863	N/A	N/A
Arthrinium morthieri	GZU 345043	MW208938	MW208864	N/A	N/A
Arthrinium phaeospermum	CBS 114317	KF144906	KF144953	KF144998	KF145040
Arthrinium phaeospermum	CBS 114318	KF144907	KF144954	KF144999	KF145041
Arthrinium puccinioides	CBS 549.86	AB220253	AB220347	AB220300	N/A
Arthrinium sphaerospermum	AP 25619	MW208943	MW208865	N/A	N/A
Arthrinium sporophleum	CBS 145154	MK014898	MK014865	MK018001	MK017973
Arthrinium trachycarpum	CFCC 53038	MK301098	N/A	MK303394	MK303396
Arthrinium trachycarpum	CFCC 53039	MK301099	N/A	MK303395	MK303397
Arthrinium urticae	IMI 326344	AB220245	AB220339	AB220292	N/A
Nigrospora aurantiaca	CGMCC 3.18130	KX986064	KX986098	KY019465	KY019295
Nigrospora camelliae-sinensis	CGMCC 3.18125	KX985986	KX986103	KY019460	KY019293
Nigrospora chinensis	CGMCC 3.18127	KX986023	KX986107	KY019462	KY019422
Nigrospora gorlenkoana	CBS 480.73	KX986048	KX986109	KY019456	KY019420
Nigrospora guilinensis	CGMCC 3.18124	KX985983	KX986113	KY019459	KY019292
Nigrospora hainanensis	CGMCC 3.18129	KX986091	KX986112	KY019464	KY019415
Nigrospora lacticolonia	CGMCC 3.18123	KX985978	KX986105	KY019458	KY019291
Nigrospora musae	CBS 319.34	MH855545	KX986110	KY019455	KY019419
Nigrospora oryzae	LC 2693	KX985944	KX986101	KY019471	KY019299
Nigrospora osmanthi	CGMCC 3.18126	KX986010	KX986106	KY019461	KY019421
Nigrospora pyriformis	CGMCC 3.18122	KX985940	KX986100	KY019457	KY019290
Nigrospora rubi	LC 2698	KX985948	KX986102	KY019475	KY019302
Nigrospora sphaerica	LC 7298	KX985937	KX986097	KY019606	KY019401
Nigrospora vesicularis	CGMCC 3.18128	KX986088	KX986099	KY019463	KY019294
Sporocadus trimorphus	CBS 114203	MH553977	MH554196	MH554636	MH554395

The ex-type cultures are indicated in bold and newly generated sequences are indicated in red. The taxa related to the Apiospora locuta-pollinis/marii clade are marked as an asterisk (*), “N/A” indicates sequence is unavailable.

Table 2

Morphological comparison among Apiospora locuta-pollinis strains.

Characteristics	Apiospora locuta-pollinis Strains
	Type Strain (LC 11683)	KUN-HKAS 124566	KUN-HKAS 124567
Host/substrate	On PDA, MEA isolated from hive-stored pollen	Decaying stem of bamboo	Dead stem of bamboo
Conidiomata	ND	150–230 high × 450–830 µm long	177–243 high × 446–682 µm long
Pycnidial wall	ND	(15–)20–40(–50) µm wide	(12–)17–40(–84) µm wide
Conidiophore mother cell	ND	(3.5–)4.5–9(–12) × 3.5–5.5 µm ($$\overline{x}$$ = 7.8 × 4.8 µm)	(4.5–)5.5–8 × (3.5–)5.5–8 µm ($$\overline{x}$$ = 5.7 × 5.5 µm)
Conidiophores	Reduced to conidiogenous cells	Pale brown, septate, branched, flexuous, (4–)5–10(–20) × (1.5–)2–3.5 µm ($$\overline{x}$$ = 8.9 × 2.2 µm)	Hyaline to light brown, septate, branched, flexuous, (8–)11–15(–24) × (1.5–)2.5–4.5 µm ($$\overline{x}$$ = 13.8 × 2.9 µm)
Conidiogenous cells	Pale brown, subglobose to ampulliform to doliiform, 3–7.5 × 3–6 μm ($$\overline{x}$$ = 4.9 × 3.8 μm)	Hyaline to light brown, cylindrical to subcylindrical or ampulliform, (3.5–)4.5–6.5(–9) × 2.5–4.5 µm ($$\overline{x}$$ = 6 × 3.5 µm)	Hyaline to light brown, subglobose to ampulliform or doliiform, (2–)3–6 × (1–)2–4.5 µm ($$\overline{x}$$ = 3.2 × 2.2 µm)
Conidia	Pale brown to brown with hyaline equatorial rim, globose to subglobose, 5.5–9 × 4.5–8 μm ($$\overline{x}$$ = 7.1 × 6.4 μm), or ellipsoidal, 8–15 × 5–9.5 μm ($$\overline{x}$$ = 10.7 × 7.1 μm)	Dark brown with a paler equatorial slit, globose to obovoid, or ellipsoidal, (8–)9–13 × (7–)9–12 µm ($$\overline{x}$$ = 10.5 × 10 µm)	Dark brown with a paler equatorial slit, globose to obovoid, or ellipsoidal, (8–)9–11 × (5.5–)8–11 µm ($$\overline{x}$$ = 9.6 × 8 µm)
Sterile cells	Pale brown or brown, ellipsoidal to clavate, 11.5–21 × 3.5–8 μm ($$\overline{x}$$ = 15.7 × 5.7 μm)	brown, leaf-like, attached to the conidiophore	ND
References	Zhao et al. [61]	This study	This study

ND = Not determined.

Table 3

Morphological comparison among Apiospora species in the A. locuta-pollinis/marii clade.

Characters	Apiospora Species
	A. marii	A. mediterranea	A. hispanica	A. gaoyouensis	A. locuta-pollinis
Host/substrate	On MEA	On MEA, isolated from Pharmaceutical excipient, atmosphere and grass	On MEA, isolated from Beach sand	On leaves and culms of Phragmites australis	On PDA, MEA isolated from hive-stored pollen
Conidiomata	NR	NR	NR	Scattered to gregarious, superficial on leaf and culms, 1–15 long × 0.5–5 mm wide	NR
Conidiophore mother cell	Ampulliform	Ampulliform	Globose to subglobose	NR	NR
Conidiophores	Basauxic, mononematous, macronematous, brownish with transverse septa of the same color	Basauxic, macronematous, mononematous, nonseptate, colorless	Basauxic, mononematous, macronematous, brownish, without transverse septa	Reduced to conidiogenous cells	Reduced to conidiogenous cells
Conidiogenous cells	NR	Integrated and terminal	NR	Aggregated in clusters on hyphae, smooth	Pale brown, smooth, subglobose to ampulliform to doliiform, 3–7.5 × 3–6 μm
Conidia	Lateral or terminal, dark brown with hyaline rim, 7.2–7.5 × 6.1–6.5 μm	Lateral or terminal, brown with hyaline rim, smooth, lenticular in shape, 9–9.5 × 7.5–9 μm	Lateral or terminal, dark brown with hyaline rim, 7.5–8.5 × 6.2–7.6 μm	Brown with pale equatorial slit, smooth, granular, globose to elongate ellipsoid in surface view, 5–8 μm diam., lenticular in side view, 4–8 μm diam., with central basal scar	Pale brown to brown with hyaline equatorial rim, smooth, globose to subglobose, 5.5–9 × 4.5–8 μm, or ellipsoidal, 8–15× 5–9.5 μm
Sterile cells	NR	Pale brown, 7–7.5 × 6.5–7 μm	Irregularly shaped, much smaller than conidia, occasionally globose	Brown, elongated cells seldom intermingled among conidia	Pale brown or brown, smooth, ellipsoidal to clavate, 11.5–21 × 3.5–8 μm
References	Larrondo and Calvo [67]	Larrondo and Calvo [68]	Larrondo and Calvo [68]	Jiang and Tian [9]	Zhao et al. [61]

NR = Not reported.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/d14110918/s1, Figure S1: Phylogenetic tree retrieved from RAxML analyses of a combined ITS, LSU and TEF1-α sequence data of Apiospora, and other related taxa in the family Apiosporaceae. Bootstrap support values for ML equal or greater than 60% and Bayesian posterior probabilities greater than 0.90 are indicated at the nodes as ML/PP. The ex-type strains are in bold. The new species are in red and new record and new combination species are in blue. The tree is rooted to Sporocadus trimorphus (CBS 114203).

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