Taxonomic and Phylogenetic Studies of Saprobic Fungi Associated with Mangifera indica in Yunnan, China

Abstract

Fungi are a large and diverse group of microorganisms, and although the estimated number of species ranges between 2 and 11 million, only around 150,000 species have been described thus far. The investigation of plant-associated fungi is beneficial for estimating global fungal diversity, for ecosystem conservation, and for the continued development of industry and agriculture. Mango, one of the world’s five most economically important fruit crops, is grown in over 100 countries and has been demonstrated to have a great economical value. During surveys of mango-associated saprobic fungi in Yunnan (China), we discovered three new species (Acremoniisimulans hongheensis, Chaenothecopsis hongheensis and Hilberina hongheensis) and five new records. The phylogenetic analyses of multi-gene sequences (LSU, SSU, ITS, rpb2, tef1-α and tub2) coupled with morphological examinations were used to identify all the taxa.

1. Introduction

Fungi prosper in various environments and play an important role in decomposition and nutrient cycling because of their ability to degrade cellulose and lignin [1,2]. A robust estimate of global fungal diversity is 2 to 11 million; however, only about 150,000 species are acknowledged to date, and tropical to subtropical areas appear to have a particularly high diversity of undiscovered fungi [3,4,5]. Fungi are distributed almost everywhere, including soil, marine water, freshwater, air, plants, animals and humans [2]. Hyde et al. [1] indicated that fungi have an enormous economic potential and listed 50 different ways that we can exploit the fungi industrially including their use in strategies against human diseases; as biocontrol agents against pests and bacterial or other fungal pathogens on plants; to enhance plant growth; for improving food and beverages; and as various commodities.

Mango is native to south Asia, India and the Malay Archipelago [6,7]. Subsequently, mango was introduced to over 100 countries, with more than 1000 varieties developed worldwide, and has always been considered an important cash crop in tropical to subtropical regions [8]. In China, the commercial cultivation of mango begun between the 1960s and 1980s and then grew rapidly, leading to China becoming the third-largest mango-producing country in the world, with 294,326 hectares of plants and 2,414,800 tons of harvested fruits in 2018 [9]. Yunnan, one of the mango-growing provinces in China, supplying 20% of the total mangoes produced nationwide, generated an economic value of CNY 1.8 billion in 2018 [10]. The main production areas in the Yunnan Province are Baoshan, Honghe, Huaping, Jinghong, Simao and Yuanjiang, and the main varieties are Keitt, Guifei, Sannian, Nang Klangwan and Jin Hwang [9].

While the investigation of plant-associated fungi contributes to the estimation of global fungal diversity, it also benefits global ecosystem conservation and industrial and agricultural development [11]. Taïbi et al. [12] mentioned that a total of 866 genera of bacteria and fungi are associated with mango, of which over 2000 species belonging to 304 genera are fungi. However, most mango-associated fungi reported earlier lack morphological details and phylogenetic supports due to limitations in the use of molecular techniques and good laboratory conditions. More recently, many mango-associated fungi have been reported along with convictive phylogenetic analysis and morphological characteristics. Recently, Tennakoon et al. [13] introduced the new species Pseudolophiostoma mangiferae and a new record of Neovaginatispora fuckelii from mango. Guo et al. [14] reported seven species of Fusarium from leaf spots of mango. Tamakaew et al. [15] established a new species of Cercosporoid fungus associated with mango in Thailand. Yang et al. [16,17] reported 20 saprobic fungi that are associated with mango in China. Therefore, many more mango-associated fungi are likely to be found in the future. In this paper, three new species and five new records of mango-associated saprobic fungi are described and introduced based on morphological comparisons and multi-gene phylogenetic analyses.

2. Materials and Methods

2.1. Sampling and Isolation

The dead and decaying woody specimens of mango were collected from rural areas of Baoshan and Honghe (Yunnan, China). The samples consisting of decayed branches, barks, and twigs (Keitt and Guifei varieties) of mango were collected and taken to the mycology laboratory. A stereo microscope (Olympus SZ61; Olympus corporation, Tokyo, Japan) was used for observing the fungal fruiting bodies on plant materials, while a digital Canon camera (EOS 600D, Canon Inc., Tokyo, Japan) fitted on to a compound microscope (Nikon ECLIPSE Ni, Nikon., Tokyo, Japan) was used to capture micro-morphological characteristics. The sizes of the main structures of fungi such as ascomata, ascomata wall, paraphyses, asci/conidiogenous cells and ascospores/conidia were measured by the Tarosoft(R) Image Frame Work program (IFW). The color photos were combined in Adobe Photoshop CS3 Extended v. 10.0 (Adobe^®, San Jose, CA, USA) to prepare complete photo plates.

Pure cultures were obtained by the single spore isolation method [18]. The spores/conidial masses were collected with a sterilized vaccination needle and gently dispersed on sterile water droplets on a micro slide. The spore suspension was transferred onto potato dextrose agar (PDA) using a micropipette and kept at 27 °C for one to two nights for germination. The single germinated spores were aseptically placed on new PDA plates, and incubated at 27 °C. The growth rate and colony characteristics of the fungal isolate were recorded after 10 days. Dried plant specimens with fungal fruiting bodies were deposited in the herbarium of the Kunming Institute of Botany Academia Sinica (HKAS), while fungal culture in tubes were deposited in the Kunming Institute of Botany Culture Collection (KUMCC). Fungal name numbers of novel species were registered as per the instruction (https://nmdc.cn/fungalnames/, accessed on 3 February 2023) [19].

2.2. DNA Extraction, PCR and Sequencing

After the culture grew for half a month, the fresh mycelium (50–100 mg) was picked up by a sterilized needle and stored in 1.5 mL centrifugal tubes for DNA extraction, for those fungal species from which we could not obtain pure cultures, approximately 5–10 fruiting bodies were carefully collected in centrifugal tubes for DNA extraction. The genomic DNA was extracted following the user instruction book of the Biospin Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux^®, Beijing, China). A part of the extracted DNA was used as template for polymerase chain reaction (PCR), while the other part was used for long-term storage at −20 °C. The 25 µL PCR mixture contained double-distilled water (8.5 µL), 2 × Power Taq PCR MasterMix (12.5 µL), DNA template (2 µL) and 2 µL of forward and reverse primers (10 pmol) (

Table 1. The PCR conditions and primers used in this study.

Locus	Definition	Primer	Annealing (PCR)	Reference
ITS	Internal transcribed spacer	ITS4	a 55 °C, 50 s b	[20]
		ITS5
LSU	Large subunit 28S	LR0R	a 55 °C, 50 s b	[21]
		LR5
rpb2	DNA-dependent RNA polymerase II largest subunit	5F	a 57 °C, 55 s b	[22]
		7cR
SSU	The partial18S small subunit	NS1	a 55 °C, 50 s b	[20]
		NS4
tef1-α	Translation elongation factor 1 alpha	983F	a 56 °C, 50 s b	[23]
		2218R
tub2	Beta-tubulin 2	Bt2a	a 52 °C, 50 s b	[24]
		Bt2b

^a Initial denaturation of 2 min at 94 °C, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing temperature and time for different genes are mentioned in Table 1. ^b 90 s of extension at 72 °C and a final extension of 10 min at 72 °C.

) [16].

The genes ITS, LSU, rpb2, SSU, tef1-α and tub2 were amplified, and the different primers used in this study are shown in Table 1. The purification and sequencing of PCR products were performed at Beijing Bio Teke Corporation.

2.3. Phylogenetic Analyses

Geneious (Restricted) 9.1.2 was used to assemble forward and reverse sequences (https://www.geneious.com, accessed on 12 May 2023), and then those combined sequences were searched in BLASTn of GenBank (http://blast.ncbi.nlm.nih.gov/, accessed on 12 May 2022) to screen relatively highly similar genera/taxa for the phylogenetic analyses (Tables S1–S8). Sequence alignments were made at the MAFFT online server version (www.ebi.ac.uk/Tools/mafft, accessed on 12 May 2022) [25] and slightly modified in BioEdit 7.2.3 [26] whenever necessary. Uninformative and unclear regions of sequences were removed by trimAL v1.2 (http://trimal.cgenomics.org, accessed on 12 May 2022), while alignments of different genes were combined in BioEdit. The online program Alignment Transformation Environment (ALTER) was used to convert Fasta files to PHYLIP (for ML) and NEXUS (for BI) formats [27]. The Maximum Likelihood analysis (ML) was run in the CIPRES Science Gateway v.3.3 (http://www.phylo.org/portal2, accessed on 12 May 2022) [28] by selecting RAxML-HPC2 on XSEDE (8.2.12) [29] and 1000 bootstrap iterations in the GTRGAMMA substitution model. The Bayesian analysis was conducted in MrBayes on XSEDE (3.2.7a) via the CIPRES Science Gateway V.3.3 web server [29,30]. Bayesian posterior probabilities (BYPP) [31,32] were assessed by Markov Chain Monte Carlo sampling (MCMC), using the best models of evolution determined by MrModeltest v. 2.3 [33] and PAUP v. 4.0b10 [34]. The GTR+I+G evolution model was also run in the BI analysis. Six simultaneous Markov chains were simultaneously subjected to Bayesian analysis for 1,000,000 to 10,000,000 generations, depending on various parameters; samples of the fungi and trees were taken, printed and produced every 1000th generation. Multi-gene phylogenetic trees were opened and checked using FigTree v1.4.0 [35], and the final trees were edited in Microsoft PowerPoint by inserting statistical supports from ML and BI.

3. Results

Taxonomic and Phylogenetic Results

Sordariomycetes O.E. Erikss & Winka 1997

Glomerellales Chadef. ex Réblová, W. Gams & Seifert, Stud. Mycol. 68: 170 (2011)

Plectosphaerellaceae W. Gams, Summerb & Zare 2007

Acremoniisimulans Tibpromma & K.D. Hyde, Fungal Diversity 93: 88 (2018)

Index Fungorum number: IF555329

Type species: Acremoniisimulans thailandensis Tibpromma & K.D. Hyde, Fungal Diversity 93: 89 (2018)

Notes: Genus Acremoniisimulans was established with the type species A. thailandensis, found associated with Pandanus sp. in Thailand, wherein only the asexual morph of this species was available [36]. Recently, Konta et al. [37] introduced a new species in this genus, a sexual morph Acremoniisimulans cocois, on dead petioles of Cocos nucifera (Arecaceae) in Thailand. To date, only above two species are accommodated in Acremoniisimulans [37]. The sexual morph of Acremoniisimulans was described as producing four or eight biseriate, oblong to broadly oblong, straight or curved, hyaline, one-celled, guttulate ascospores in each oblong to clavate, and pedicellate ascus; Acremoniisimulans asexually described by having solitary, hyaline or subhyaline to pale brown, oval, aseptate, slimy, conidia formed on macronematous, mononematous, scattered, smooth, and thick-walled conidiophores [36,37]. The multi-gene phylogenetic placements of Acremoniisimulans sp. and other genera in Plectosphaerellaceae are shown in Figure 1.

Acremoniisimulans hongheensis E.F. Yang & Tibpromma, sp. nov. (Figure 2)

Fungal Name number: FN571274

Etymology: The name reflects the location, Honghe, from where the holotype was collected.

Holotype: HKAS 122669

Saprobic on dead twigs of Mangifera indica. Sexual morph: Visible as numerous black, raised dots beneath the epidermis of the host. Ascomata (excluding neck) 120–150 μm × 120–195 μm ($\overline{\mathrm{x}}$ = 140 × 160 μm, n = 10) diam., immersed, globose to subglobose, exposed when horizontally sectioned through bark surface, scattered to gregarious, with a short ostiole. Ostiole 50–75 μm × 50–75 μm ($\overline{\mathrm{x}}$ = 65 × 60 μm, n = 10) diam., central, cylindrical to narrowly canalled, with darkly pigmented cells in the neck. Ascomata wall 15–25 μm ($\overline{\mathrm{x}}$ = 19 μm, n = 20) wide, multilayered, with inner layers comprising hyaline, long and narrow cells of textura prismatica, outer layers formed with thick-walled cells of textura angularis. Hamathecium composed of 2–5 μm ($\overline{\mathrm{x}}$ = 4 μm, n = 20) wide, filamentous, dense, cylindrical, septate, unbranched, straight to slightly curved, paraphyses formed from a gelatinous matrix. Asci 40–50 × 5–7 μm ($\overline{\mathrm{x}}$ = 46 × 6 μm, n = 10), eight spored, bitunicate, oblong, cylindrical, hyaline, short pedicellate, apically rounded, with a vague apical chamber. Ascospores 5–8 × 2–4 μm ($\overline{\mathrm{x}}$ = 6.5 × 3 μm n = 20), uni- or bi-seriately overlapping, ellipsoidal, septate, obtuse at both ends, thick and smooth walled, without a gelatinous sheath or appendages. Asexual morph: Undetermined.

Material examined: China, Yunnan Province, Baoshan City, Longling County, on dead twigs of Mangifera indica (99°16′80″ E, 25°12′23″ N, Elevation: 800 m) 27 December 2019, E.F. Yang, erfu1 (Herb. HKAS 122669, holotype); isotype HKAS 122670. GenBank numbers: HKAS 122669 = ITS: OQ379005, LSU: OQ379416, SSU: OQ372921, tef1-α: OQ378995, rpb2: OQ378988; HKAS 122670 = ITS: OQ379006, LSU: OQ379417, SSU: OQ372922, tef1-α: OQ378996, rpb2: OQ378989.

Notes: Based on morphology, our isolate shares similar sexual morph with Acremoniisimulans cocois (Plectosphaerellaceae), by producing biseriate asci, oblong to ellipsoidal, hyaline, 1-celled, guttulate, thick and smooth-walled ascospores and without a gelatinous sheath or appendages [37]. In addition, the BLASTn results of LSU and SSU showed 97–99% similarity with the Acremoniisimulans thailandensis (MFLUCC 16-0372) and A. cocois (MFLUCC 15-0817). However, the BLASTn results of ITS and tef-α showed a low similarity (91%) with Verticillium isaacii (CBS100839), V. tricorpus (CBS100867) and Chlamydosporiella restricta (CBS 178.40, CBS177.40); in addition, the rpb2 gene even has a similarity of only 79% with Acremonium collariferum (CBS124585). The multi-gene phylogenetic trees also indicated that Acremoniisimulans hongheensis strains separated from A. cocois and A. thailandensis (Figure 1). Therefore, Acremoniisimulans hongheensis is established as the third species in the genus Acremoniisimulans [37].

Dothideomycetes sensu O.E. Erikss & Winka

Tubeufiales Boonmee & K.D. Hyde, in Boonmee, Rossman, Liu, Li, Dai, Bhat, Gareth Jones, McKenzie, Xu & Hyde, Fungal Diversity 68(1): 245 (2014)

Tubeufiaceae M.E. Barr, Mycologia 71(5): 948 (1979)

Excipulariopsis P.M. Kirk & Spooner, in Spooner & Kirk, Trans. Br. mycol. Soc. 78(2): 251 (1982)

Index Fungorum Registration Identifier: IF8228

Type species: Excipulariopsis narsapurensis (Subram.) Spooner & P.M. Kirk 1982

Notes: The family Tubeufiaceae contains about 47 genera and more than 400 species [38]. The monotypic genus Excipulariopsis was established with E. narsapurensis as the type species [39], and the asexual morph has raised stroma, cylindrical, hyaline, holoblastic conidiogenous cells and fusiform, transversely multi-septate, brown, verruculose, acrogenous conidia with a truncate base [39,40]. However, molecular data for Excipulariopsis narsapurensis are unavailable, and therefore, the phylogenetic placement remained unsettled until this study. Multi-gene BLASTn (ITS, LSU, tef1-α and rpb2) searchers of our isolate remained relatively low in similarity with the strains of other genera. The BLASTn searches of ITS and tef1-α of our isolates (KUMCC 21-0464, 21-0465) showed a low similarity (85–87%) with Chlamydotubeufia khunkornensis (MFLUCC 10-0117), Tubeufiaceae sp. (MFLUCC 16-1129), Helicosporium flavum (MFLUCC 16-1230) and Tubeufia tectonae (MFLUCC 17-1985); the LSU gene has a similarity of 96% to Parawiesneriomyces chiayiensis (MFLUCC 20-0041) and Thaxteriellopsis lignicola (MFLUCC 16-0026); and the rpb2 gene has a similarity of 80% to Berkleasmium latisporum (MFLUCC 16-0019) and Helicoma multiseptatum (GZCC 16-0080). The multi-genetic phylogenetic trees (ML and BI) formed a well-supported subclade close to genus Thaxteriellopsis with low statistical support (66% in ML, 0.99 in BI; Figure 3), but they were well separated from Neotubeufia and Thaxteriellopsis. Therefore, this study showed the phylogenetic placement of the genus Excipulariopsis for the first time (Figure 3).

Excipulariopsis narsapurensis (Subram.) Spooner & P.M. Kirk, Trans. Br. mycol. Soc. 78(2): 251 (1982) (Figure 4)

=Excipularia narsapurensis Subram., J. Indian bot. Soc. 35(1): 56 (1956)

Index Fungorum Registration Identifier: IF110673

Saprobic on a dead bark of Mangifera indica. Sexual morph: Unknown. Asexual morph: Hyphomycetous. Colonies gregarious, effuse, superficial, pulvinate, setiferous, sporodochial, recognized as shining black regions, distinctly scattered on the surface of the host, with erect, 7–10 μm-wide setae. Stroma composed of dark, thick, brown-walled stromatic cells. Setae erect, straight, septate, tubular, black, peripheral, smooth, with acute ends. Conidiogenous cells 12–20 × 4–6 μm ($\overline{\mathrm{x}}$ = 16 × 5 μm), holoblastic, cylindrical, hyaline to lightly brown, smooth walled, straight to slightly flexuous, arise on stroma as a palisade layer. Conidia 40–55 × 9–30 μm ($\overline{\mathrm{x}}$ = 48 × 19 μm n = 20), 4–7 septate, acrogenous, fusiform, markedly constricted at the septum, smooth walled, apically rounded at both ends, granulate, thick walled, brown to dark brown, hyaline to pale brown at both end cells, with thick and dark bands at septa, often carrying a small part of conidiogenous cell at the base.

Culture characteristics: Conidia produced germ tubes from both ends within 24 h at 27 °C, rapidly growing, colony up to 20–30 mm after one week; obverse: circular, flat or effuse, crenated at the edge, white to gray pigments at the center, with medium-level density; reverse: Brown to dark brown from outer to inner parts, pale brown at the margin, without pigments produced in PDA.

Substratum: On decaying wood with corticeaceous fungus [39,40]; Cocos nucifera [41], the bark of Mangifera indica (this study).

Distribution: Hawaii, USA [39,40]; Dapoli, India [41]; Yunnan Province, China (this study).

Material examined: China, Yunnan Province, Honghe, Menglong Village, on a dead and decaying bark of Mangifera indica (102°50′11″ E, 23°41′01″ N, 500 m), 22 December 2020, E.F. Yang, HHE018 (HKAS 122680), living culture, KUMCC 21-0464 = KUMCC 21-0465. Genbank number: KUMCC 21-0464 = ITS: OQ379007, LSU: OQ379418, tef1-α: OQ378997, rpb2: OQ378990;KUMCC 21-0465 = ITS: OQ379008, LSU: OQ379419, tef1-α: OQ378998, rpb2: OQ378991.

Notes: The asexual morphs of Tubeufiaceae are hyphomycetous, often with helicosporous, phragmosporous or sometimes chlamydosporous conidia [42,43]. In this study, our collection resembled Excipulariopsis narsapurensis (BISH 594584) in having effuse, superficial, sporodochial colonies, with cylindrical, hyaline, holoblastic conidiogenous cells and brown, phragmoseptate, acrogenous conidia (68–72 × 23–27 µm vs. 40–55 × 9–30 μm [40]. Multi-gene BLASTn (ITS, tef1-α and rpb2) searchers showed relatively low similarity (<90%) with strains of other genera (e.g., Chamydotubeufia, Thaxteriellopsis, Tubefufia and Neoacanthostigma). Based on multi-gene analyses of LSU, tef1-α, rpb2 and ITS datasets, strains of Excipulariopsis narsapurensis are seen as clearly distinguishable from other generic species in the Tubeufiaceae. Therefore, in this study, with the phylogenetic placements and molecular data, we propose a new geographic and a new host record, for Excipulariopsisnarsapurensis.

Eurotiomycetes Eurotiomycetes O.E. Erikss & Winka, Myconet 1: 6 (1997)

Mycocaliciales Tibell & Wedin, Mycologia 92(3): 579 (2000)

Mycocaliciaceae A.F.W. Schmidt, Mitt. Staatsinst. Allg. Bot. Hamburg 13: 127 (1970)

Chaenothecopsis Vain., Acta Societatis pro Fauna et Flora Fennica 57 (1): 70 (1927)

Index Fungorum Registration Identifier: IF934

Type species: Chaenothecopsis rubescens Vain., Acta Societatis pro Fauna et Flora Fennica 57: 71 (1927)

Notes: Chaenothecopsis is a genus of the ascomycetes in the order Mycocaliciales [44], and to date, it accommodates a total of 101 records in Index Fungorum (2023) [45]. The majority of Chaenothecopsis taxa were previously known as resinicolous mycocalicioid taxa, which are saprotrophic on wood, resin (resinicolous), or associated with lichens as green algal symbionts [46,47]. In addition, Chaenothecopsis polissica as a fossil fungus was found in Rovno amber [48]. The taxa of this genus have raised, dark brown, branched or unbranched, straight to curved, apically fertile, broad apothecioid, synnematous fructifications. The sexual morph of Chaenothecopsis has oval to ellipsoidal, normally brown, ascospores with or without septa, and it is wrapped in slender cylindrical asci, while the asexual morph has rarely been reported before. Asexual morph fruiting bodies of Chaenothecopsis species are synnematous, with straight, parallel conidiophores compacted in the upper fertile parts, cylindrical, terminal or intercalary, tiny conidiogenous cells, and obovoid to clavate, aseptate, verruculose, catenated, olivaceous to brown conidia [49,50]. In this study, we provide the phylogenetic tree of Chaenothecopsis with their closely related groups (Figure 5).

Chaenothecopsis hongheensis E.F. Yang & Tibpromma, sp. nov. (Figure 6).

Fungal Name number: FN571288

Etymology: The name reflects the location, Honghe, from where the holotype was collected.

Holotype: HKAS 122672

Saprobic on resin of dead branch of Mangifera indica. Sexual morph: Undetermined. Asexual morph: Synnemata erect on exudate or substrate, 380–470 μm ($\overline{\mathrm{x}}$ = 420 μm, n = 10) high in total, 100–270 μm ($\overline{\mathrm{x}}$ = 420 μm, n = 10) diam. at apical parts, dark grayish brown, straight or sometimes slightly flexuous, with obovoid to lentil-shaped head. Stipe 250–370 μm ($\overline{\mathrm{x}}$ = 300 μm, n = 10) high, 60–120 μm ($\overline{\mathrm{x}}$ = 90 μm, n = 10) wide, grayish black, relatively short, rarely branched, thickened at the bottom, composed of parallelly adpressed conidiophores. Conidiophores 45–65 μm ($\overline{\mathrm{x}}$ = 55 μm, n = 20) high, 2–3 μm ($\overline{\mathrm{x}}$ = 2.5 μm, n = 20) wide, compacted below, slightly flared on capitulum, numerous, sometimes apically branched, septate, flexuous, pale brown to brown with the maturity. Conidiogenous cells 1–2 μm wide, 2–3 μm long, lateral, polyblastic, terminal or intercalary, hyaline, short cannular to cylindrical, smooth. Conidia 4–5.5 × 2.5–4 μm ($\overline{\mathrm{x}}$ = 4.5 × 3.5 μm, n = 20), obovoid to clavate, aseptate, flat to rounded at apex, verruculose, olivaceous to brown, rough walled, catenate, without a mucilaginous sheath.

Material examined: China, Yunnan Province, Honghe, Menglong Village, on the resin of a dead branch of Mangifera indica (102°50′11″ E, 23°41′01″ N, 500 m), 22 December 2020, E.F. Yang, erfu12 (Herb. HKAS 122673, holotype); isotype HKAS 122672. GenBank numbers: HKAS 122672 = ITS: OQ379009, LSU: OQ379420; HKAS 122673 = ITS: OQ379010, LSU: OQ379421.

Notes: In this study, we established a new resinicolous species of Chaenothecopsis growing on the resin of Mangifera indica. Our isolate fits very well within the common concept of Chaenothecopsis, with brown to dark brown, solitary, apothecioid, fructification emerging near to resin. However, only the asexual morph of our new isolate was available as synnematous with visible, erect, sometimes branched conidiophores on apical parts, conidiogenous cells lateral, terminal or intercalary, short granular, indistinct; and ovoid to obpyriform, olivaceous to brown, verruculose conidia. The asexual morph characteristics are distinguishable from other taxa in the Chaenothecopsis [53]. Additionally, the phylogenetic analyses also indicated that Chaenothecopsis hongheensis strains (HKAS 122672, 122673) are related to C. pallida (H: JR 10652) and C. quintralis (BCRU: 05233). A complete comparison of the 789 nucleotides of the LSU gene region from C. pallida (H: JR 10652) with our isolates revealed 26 base pair differences (3.29%) with 0 gaps. A detailed comparison of the 514 nucleotides of the ITS gene region from our isolate and C. pallida (H: JR 10652) revealed 68 base pair differences with 29 gaps. As our isolate is well separated from Chaenothecopsis pallida (H: JR 10652) and C. quintralis (BCRU: 05233) in morphology and phylogeny, we introduce C. hongheensis as a distinct new species with a unique anamorph.

Sordariomycetes O.E. Erikss & Winka 1997

Sordariales Chadef. ex D. Hawksw & O.E. Erikss., Syst. Ascom. 5(1): 182 (1986)

Helminthosphaeriaceae Samuels, Cand &Magni 1997

Hilberina Huhndorf & A.N. Mill., Mycological Research 108 (1): 31 (2004)

Index Fungorum Registration Identifier: IF28830

Type species: Hilberina caudata (Fuckel) Huhndorf & A.N. Mill., Mycological Research 108 (1): 31 (2004)

Notes: The genus Hilberina was re-described by Miller and Huhndorf [54,55], with H. caudata as the type species. The sexual morphological characteristics of this genus are as follows: Often associated with dead woody materials, presenting superficial, subglobose to ovoid ascomata with thick walled, brown, long setae and papillate ostiole; oblong, cylindrical asci contain subglobose, septate or aseptate, hyaline ascospores without gelatinous appendages. Molecular data are available for only five species (Hilberina caudata, H. sphagnorum, H. punctata, H. robusta and H. munkii). The members of Hilberina are very closely related to Synaptospora, Ruzenia and Helminthosphaeria (Helminthosphaeriaceae) in morphological characteristics and phylogenetic placements [56,57]. To date, 20 species records of Hilberina are in Species Fungorum (2023) [58], and many of them were transferred from Lasiosphaeria and Sphaeria based on morphology [56]. One species of Hilberina (H. breviseta) associated with submerged wood in Dianchi Lake has been reported from Yunnan Province, China [59]. In this study, we updated the multi-gene phylogenetic tree of Hilberina as shown in Figure 7.

Hilberina hongheensis E.F. Yang & Tibpromma, sp. nov. (Figure 8)

Fungal Name number: FN571289

Etymology: The name reflects the location, Honghe, from where the holotype was collected.

Holotype: HKAS 122677

Saprobic on a dead branch of Mangifera indica. Sexual morph: Ascomata 135–160 × 145–180 μm ($\overline{\mathrm{x}}$ = 150 × 160 μm, n = 10), superficial, subglobose, scattered, solitary, carbonaceous black, setiferous on the surface of the substrate, with long black papilla visible at the surface, with a poorly developed ostiole. Setae 7–10 high, pale brown to brown, sub-hyaline at the tip, septate, straight to slightly flexuous, smooth, with a narrow acute apex. Ascomata wall 25–35 ($\overline{\mathrm{x}}$ = 31 μm, n = 10) μm wide, dark brown, multilayered, comprising hyaline cells of textura angularis inwardly, with outer layers appearing dark brown and thick walled. Hamathecium composed of 2–5 μm-wide, septate, cylindrical, filamentous, unbranched paraphyses constricted at septa. Asci 70–85 × 7–10 μm ($\overline{\mathrm{x}}$ = 75 × 9 μm, n = 20), eight spored, unitunicate, often four spored when immature, well-developed when mature, cylindrical, oblong, hyaline, slightly shining, short pedicellate, apically rounded, thick walled; sometimes with four poorly developed ascospores in each ascus. Ascospores 13–16 × 6–9 μm ($\overline{\mathrm{x}}$ = 15 × 8 μm, n = 30), uniseriately overlapping, oval, hyaline, thin and rough walled, verruculose, with oil droplets disappearing on maturity, without a gelatinous sheath and appendages. Asexual morph: Undetermined.

Material examined: China, Yunnan Province, Baoshan City, Longling County, on a dead and decaying branch of Mangifera indica (99°16′80″ E, 25°12′23″ N, Elevation: 800 m) 27 December 2019, E.F. Yang, MB004 (HKAS 122677, holotype); isotype HKAS 122678, GenBank numbers: HKAS 122677 = LSU: OQ379422, tub2: OQ379003; HKAS 122678 = LSU: OQ379423, tub2: OQ379004, tef1-α: OQ379002.

Notes: Based on morphology, our isolate fits well within the generic concepts of Hilberina rather than Helminthosphaeria and closely resembles the type species of Hilberina, (H. caudata). However, H. caudata was originally described as having ascomata with setae, papillate neck; cylindrical, eight-spored asci with a refractive, inamyloid apical ring; septate, cylindrical, geniculate, hyaline to pale brown ascospores with one end tapering to a thin tip [55,60]. Our new isolate differs from H. caudata by subglobose to broadly fusiform, one-celled ascospores. The BLASTn search results and percent sequence of LSU and tub2 show similarity to H. caudata strains SMH 1542 with 95.8% and 98.7%, respectively. Consequently, our isolates are identified as a distinct novel species in Hilberina based on morphological and phylogenetic studies.

Sordariomycetes O.E. Erikss & Winka 1997

Delonicicolales R.H. Perera, Maharachch & K.D. Hyde 2017

Delonicicolaceae R.H. Perera, Maharachch & K.D. Hyde, in Perera, Maharachchikumbura, Jones, Bahkali, Elgorban, Liu, Liu & Hyde, Cryptog. Mycol. 38(3): 334 (2017)

Delonicicola R.H. Perera, Maharachch & K.D. Hyde, Cryptogamie, Mycologie 38: 334 (2017).

Index Fungorum Registration Identifier: IF553771

Type species: Delonicicola siamense R.H. Perera, Maharachch. & K.D. Hyde, Cryptogamie, Mycologie 38: 335 (2017)

Notes: The genus Delonicicola was first established for a sexual morph, with D. siamense as the type species. The type species was found as saprobic on dried seed pods of Delonix regia in Thailand, and full descriptions and morphological characteristics were offered by Perera et al. [61]. Currently, Delonicicola is placed under the family Delonicicolaceae along with Liberomyces and Furfurella [62,63]. The sexual morph of Delonicicola was introduced as having grouped, black, conspicuous, raised pseudo-stromata, with multi-loculate, aggregated, immersed, globose to conical, ostiolate, ascomata; paraphyses filamentous, aseptate, unbranched; eight-spored, unitunicate, clavate, short pedicellate asci; ascospores are one or two seriate, one septate, blunt at ends, straight, hyaline, and smooth walled [61]. The asexual morph of Delonicicola has so far not been reported and herein introduced for the first time. Conidiomata immersed, multi-loculate, with a prolonged neck; Conidiophores aseptate, cylindrical, straight to slightly curved, sometimes branched. Conidiogenous cells holoblastic, with a cylindrical lumen. Conidia lunate, zero-to-one septate, hyaline, curved, and tapering towards apex. The phylogenic relationships of Delonicicola (Delonicicolaceae) and closely related genera/families are shown in Figure 9.

Delonicicola siamense R.H. Perera, Maharachch & K.D. Hyde, Cryptogamie, Mycologie 38: 335 (2017) (Figure 10)

Index Fungorum Registration Identifier: IF553771

Saprobic on dead stem of Mangifera indica. Sexual morph: Available in Perera et al. [61]. Asexual morph: Conidiomata 190–340 × 120–170 μm ($\overline{\mathrm{x}}$ = 265 × 150 μm, n = 20), pycnidial, fully immersed in plant tissues, with apical part of prolonged neck visible above the surface, multi-loculate, conspicuous at surface, dark brown. Conidiomata wall 11–17 μm ($\overline{\mathrm{x}}$ = 14 μm, n = 20), comprising thick-walled, brown cells of textura epidermoidea, 3.5–6.5 μm ($\overline{\mathrm{x}}$ = 5 μm, n = 20) wide at outer layers, merged with the host. Paraphyses absent. Conidiophores 15–30 μm ($\overline{\mathrm{x}}$ = 18 μm, n = 20) high, 1–2 μm ($\overline{\mathrm{x}}$ = 1.5 μm, n = 20) wide, hyaline, branched, septate, cylindrical, straight to slightly flexuous. Conidiogenous cells 1–2 μm high, 0.5–1 μm wide, nodose at the tip, polyblastic, sympodial, slightly bulged at the apex. Conidia 19–26 × 1.5–3 μm ($\overline{\mathrm{x}}$ = 22 × 2.5 μm, n = 20), lunate, zero-to-one septate, with a central septum, granulate, smooth, truncate at base, curved above half and tapering to apex.

Culture characters: Conidia formed germ tube on PDA within 24 h at room temperature, fast-growing and colonies reaching around 30 mm within half a month, colonies circular, flat to effuse, slightly radiating, gray to whitish outwardly, with entire margin; reverse: reddish brown at the center, becoming white towards margin. Without pigments released in PDA.

Substratum: Seed pods of Delonix regia [61]; a dead stem of mango (Mangifera indica) (this study).

Distribution: Chiang Rai, Thailand [61]; Yunnan, China (this study).

Material examined: China, Yunnan Province, Honghe, Menglong Village, on a dead stem of Mangifera indica (102°50′11″ E, 23°41′01″ N, 500 m), 22 December 2020, E.F. Yang, MB012-1 (Herb. HKAS 122662); living culture KUMCC 21-0459. GenBank numbers: KUMCC 21-0459 = ITS: OQ379013, LSU: OQ379424, rpb2: OQ378992.

Notes: The genus Delonicicola, with D. siamense as the type species, was introduced by Perera et al. [61], and it is so far known only from its sexual morph. Multigene phylogenetic analysis (ML and BI) showed our isolate (KUMCC 21-0459) clusters with D. siamense (MFLUCC 15-0670) with high statistical support (100% in ML, 1 in BI; Figure 9). Moreover, the BLASTn results of ITS, LSU and rpb2 indicated that our isolate belongs to Delonicicola siamense with a similarity of >99%. Morphologically, the conidia of D. siamense (KUMCC 21-0459) are zero-to-one septate at the central, hyaline, granulate and slightly curved. Our isolate (KUMCC 21-0459) is identified as Delonicicola siamense based on morphological examinations together with phylogenetic analyses. In addition, this is the first report of an asexual morph of D. siamense and a new host and country record.

Sordariomycetes O.E. Erikss & Winka 1997

Coronophorales Nannf., Nova Acta R. Soc. Scient. upsal., Ser. 4 8(no. 2): 54 (1932)

Nitschkiaceae Nannf., Nova Acta R. Soc. Scient. upsal., Ser. 4 8(no. 2): 56 (1932)

Fracchiaea Sacc., Nuovo Giornale Botanico Italiano 5: 285 (1873)

Index Fungorum Registration Identifier: IF2008

Type species: Fracchiaea heterogenea Sacc., Nuovo Giornale Botanico Italiano 5: 285 (1873)

Notes: Fracchiaea, as typified by F. heterogenea, is nested in the Nitschkiaceae [64], and the genus is basal to the clade of Acanthonitschkea and Nitschkia in the phylogeny (Figure 11). This genus includes 35 species, but only three species (Fracchiaea broomeana, F. lunata, and F. myricoides) have molecular data [65,66]. The species of Fracchiaea are commonly saprobic on wood. The sexual morph of Fracchiaea differs by being immersed to erumpent, carbonaceous to coriaceous, black, turbinate ascomata; polysporous, unitunicate, clavate to cylindrical, pedicellate or sessile asci; hyaline to yellowish, ellipsoidal to cylindrical to allantoid, slightly flexuous, numerous, zero-to-one septate ascospores. However, asexual morph is undetermined [65,66].

Fracchiaea myricoides (H.X. Wu & K.D. Hyde) S.K. Huang & K.D. Hyde, Mycosphere 12 (1): 920 (2021) (Figure 12)

Index Fungorum Registration Identifier: IF558200

Saprobic on a dead bark of Mangifera indica. Sexual morph: Ascomata up to 280–330 × 310–420 μm ($\overline{\mathrm{x}}$ = 275 × 365 μm, n = 10), perithecial, clustered in small to large groups, superficial or erumpent, globose to subglobose, with poorly developed ostiole at the center, rough at the surface, tuberculate, dark brown to carbonaceous black. Ascomata wall 45–70 μm wide, multilayered, thick walled, composed of multilayered dark brown-walled cells of textura globosa; rough at the outer surface, comprised of hyaline pseudoparenchymatic cells of textura angularis in inner layers 15–55 μm ($\overline{\mathrm{x}}$ = 35 μm, n = 20). Paraphyses absent. Asci 100–150 × 12–26 μm ($\overline{\mathrm{x}}$ = 125 × 19 μm, n = 20), numerous, unitunicate, polysporous, oblong, cylindric clavate, short pedicellate, round or blunt at apex, without a visible discharge mechanism. Ascospores 6–8 × 1.5–2 μm ($\overline{\mathrm{x}}$ = 7 × 2 μm), numerous, crowded, hyaline, cylindrical to allantoid, slightly curved, aseptate, smooth walled, with granules. Asexual morph: Undetermined.

Substratum: Dead wood of an unidentified plant [67] dead mango bark (this study).

Distribution: Yunnan Province, China ([67], this study).

Material examined: China, Yunnan Province, Honghe, Menglong Village, on a dead bark of Mangifera indica (102°50′11″ E, 23°41′01″ N, 500 m), 22 December 2020, E.F.Yang, HHE020 (Herb. HKAS 122671). GenBank numbers: HKAS 122671 = ITS: OQ379014, LSU: OQ379425, tef1-α: OQ378999, rpb2: OQ378993.

Notes: Our isolate fits well within the concept of Fracchiaea by forming gregarious, carbonaceous to coriaceous, black ascomata without ostiole on a woody surface; ascomata wall comprising textura angularis to textura prismatica cells; without paraphyses; zero-to-one septate, numerous ascospores wrapped in a clavate-to-cylindrical ascus. Based on multi-gene phylogenetic analysis of combined LSU, tef1-α and rpb2 sequence data, our isolate (HKAS 122671) is well clustered with the ex-type strain of F. myricoides (IFRD 9201) with high and reliable statistical support (Figure 11). In addition, our isolate and F. myricoides (IFRD 9201) have similarly sized ascomata, asci and ascospores (ascomata: $\overline{\mathrm{x}}$ = 275 × 365 μm vs. $\overline{\mathrm{x}}$ = 383 × 308 μm; asci: $\overline{\mathrm{x}}$ = 125 × 19 μm vs. $\overline{\mathrm{x}}$ = 119 × 22 μm; ascospore: $\overline{\mathrm{x}}$ = 7 × 2 μm vs. $\overline{\mathrm{x}}$ = 7.9 × 1.7 μm) [66,67]. In addition, in mega BLASTn search using the LSU sequence, the closest matches of F. myricoides (IFRD 9201) in NCBI’s GenBank nucleotide database showed 99.06% similarity (Identities = 951/960 bp; Gaps = 0). Unfortunately, the tef1-α and rpb2 molecular data of Fracchiaea myricoides (IFRD 9201) were unavailable. Nevertheless, we regard our isolate as F. myricoides, and this is the first time to report F. myricoides from Mangifera indica based on morphological examination and phylogenetic analysis. In addition, Coronophora myricoides originally collected from unidentified woody debris in China has been transferred to the Fracchiaea based on phylogenetic analysis and morphological comparison [66].

Dothideomycetes sensu O.E. Erikss & Winka 1997

Monoblastiales Lücking, M.P. Nelsen & K.D. Hyde, in Hyde et al., Fungal Diversity 63: 8 (2013)

Monoblastiaceae Walt. Watson, New Phytol. 28: 106 (1929)

Heleiosa Kohlm., Volkm.-Kohlm & O.E. Erikss., Can. J. Bot. 74: 1830 (1996)

Index Fungorum Registration Identifier: IF27767

Type species: Heleiosa barbatula Kohlm., Volkm.-Kohlm & O.E. Erikss. 1996.

Notes: The monotypic genus Heleiosa was established by Kohlmeyer in 1996 [68], with H. barbatula as the type species. Heleiosa barbatula was collected from Junciroemeriani in the USA (North Carolina). The morphological characteristics of this genus differ by immersed, papillate, clypeate ascomata, without neck, sparse periphyses around ostiolar canal; ascomata wall is light colored, thin, comprising textura angularis cells; pseudoparaphyses branched, anastomosis, extending over the asci; asci are four-to-eight spored; producing fusiform, brown, one-celled ascospores, with numerous cilia-like sub-apical appendages at each end [68]. Lately, the phylogenetic analysis also supported Heleiosa as a separate genus in the Monoblastiaceae. It has been speculated that H. barbatula and Funbolia dimorpha, as non-lichen-forming taxa, strongly support a close relationship with the Monoblastiaceae and that they could provide insight into the evolution of the lichen-forming habit [69,70]. Moreover, Neoheleiosa was introduced close to Heleiosa (type strain: JK5548I) with high statistical support in phylogenetic trees [71]. Heleiosa is still a poorly studied group, and only one single species with a sexual morph is available. The phylogeny of Heleiosa and relative genera is shown in Figure 13.

Heleiosabarbatula Kohlm., Volkm.-Kohlm & O.E. Erikss., Canadian Journal of Botany 74: 1830 (1996) (Figure 14)

Index Fungorum Registration Identifier: IF436638

Saprobic on a dead branch of Mangifera indica. Sexual morph: Ascomata (excluding ostiole) 180–280 × 200–280 μm ($\overline{\mathrm{x}}$ = 230 × 240 μm, n = 20), globose to ampulliform, brown to dark brown, aggregated, sometimes solitary, uni- or multiloculate, fully immersed beneath host epidermis when immature, raised with erumpent neck when mature, visible as black scars on the surface. Ostiole 45–60 × 100–125 μm ($\overline{\mathrm{x}}$ = 50 × 115 µm, n = 20), brown, central, cylindrical, flat at the apex. Perdium 25–40 μm ($\overline{\mathrm{x}}$ = 34 µm, n = 20) wide, multilayered, hyaline to lightly brown comprising hyaline cells of textura prismatica in inner layers, thick walled, hyaline to lightly brown, outermost cells merged with plant tissues. Hamathecium generated from a gelatinous matrix, composed of 1–2 µm-wide, filamentous, hyaline, numerous, branched, septate, pseudo-paraphyses. Asci 100–130 × 8–13 µm ($\overline{\mathrm{x}}$ = 120 × 11 µm, n = 30), eight spored, bitunicate, short pedicellate, cylindrical, with a furcated base, apically rounded with an indistinct ocular chamber. Ascospores 14–18 × 6–9 µm ($\overline{\mathrm{x}}$ = 15 × 8 µm, n = 30), uniseriate, one septate, ellipsoidal, slightly constricted at the septum, hyaline to green-brown, with oil droplets, obtuse ends, smaller upper cell, thick walled.

Culture characteristics: On PDA media, ascospores randomly produced germ tubes near obtuse ends within 20 h, taking two months to develop 10 mm-diam. Colonies at room temperature slowly; colonies circular, fluffy, convex, brown, dense; reverse: dark reddish brown, white at the margin, without pigments produced in PDA.

Substratum: Rush of Junciroemeriani [68]; dead branch of Mangifera indica (this study).

Distribution: North Carolina, Virginia, the USA [68]; Yunnan Province, China (this study).

Material examined: China, Yunnan Province, Baoshan City, Longling County, on a dead branch of Mangifera indica (99°16′80″ E, 25°12′23″ N, Elevation: 800 m) 27 December 2019, E.F. Yang, MB012 (HKAS 122668), living culture, KUMCC 21-0462. GenBank number: HKAS 122668 = ITS: OQ379015, LSU: OQ379426, SSU: OQ372923, tef1-α: OQ379000.

Notes: Heleiosa has only one species (H. barbatula) as described by Kohlmeyer et al. [68]. Our isolate fits well within the concept of Heleiosa barbatula by producing immersed, subglobose ascomata; asci are oblong, with an ocular chamber at the apex, and have a similar size (75–100 × 9–11 µm vs. 100–130 × 8–13 µm); ascospores are ellipsoidal to cylindrical, one septate, with obtuse ends, green brown to brown, with oil globules, and have a similar size (16.5–22.5 (–24) × 5.5–7 µm vs. 14–18 × 6–9 µm). In addition, a comparison of the LSU region of our isolate (KUMCC 21-0462) and Heleiosa barbatula (JK 5548I) reveals 98% similarity, and the SSU region reveals 100% similarity. Although Heleiosa barbatula (JK 5548I) lacks ITS and tef1-α sequencing data, the morphological characteristics and multi-gene phylogenetic results fully support our isolate as the same species as H. barbatula (Figure 13). Therefore, we report Heleiosa barbatula (KUMCC 21-0462) on Mangifera indica as a new host and country record.

Dothideomycetes sensu O.E. Erikss & Winka 1997

Pleosporales Luttr. ex M.E. Barr, Prodr. Cl. Loculoasc. (Amherst): 67 (1987)

Hermatomycetaceae Locq., Mycol. gén. struct. (Paris): 202 (1984)

Hermatomyces Speg., Anales del Museo Nacional de Historia Natural Buenos Aires ser. 3, 13: 445 (1911)

Index Fungorum Registration Identifier: IF8517

Type species: Hermatomyces tucumanensis Speg., Anales del Museo Nacional de Historia Natural Buenos Aires ser. 3, 13: 446 (1911)

Notes: Speggazini (1911) [72] established the genus Hermatomyces on the basis of material collected in Argentina, with H. tucumanensis as the type species. Speggazini [72] described only lenticular-type conidia and considered the cylindrical ones as conidiophores. However, with further morphological and phylogenetic investigations, the muriform, lenticular, hyaline or dematiaceous conidia of some Hermatomyces sp. were reported (previously referred to as monomorphic or dimorphic), and different species were mostly distinguished by cylindrical conidia [73,74]. Species of Hermatomyces seem to be limited by elevation and climate, and prefer to live on humid plant materials, including those found on immersed woody materials in water [73,75]. The ability of cylindrical conidia to germinate has not been proved; Koukol et al. [73] speculated that the function of cylindrical conidia is to support the lenticular conidia, and does not contribute to reproduction. To date, this genus includes a total of 34 records in Species Fungorum (2023) [58]. The phylogenetic placements of Hermatomyces species are shown in Figure 15.

Hermatomyces indicus Prasher & Sushma, Nova Hedwigia 99: 552 (2014) (Figure 16)

Index Fungorum Registration Identifier: IF805645

Saprobic on dead decaying branch of Mangifera indica. Sexual morph: Undetermined. Asexual morph: Colonies formed a dull zone on natural substrate, scattered to gregarious, in several groups, forming circular brownish mycelial outer region with abundantly sporulating center, with conidia easily liberated when touched with a needle. Mycelium 2–3 ($\overline{\mathrm{x}}$ = 2.5 μm, n = 20) wide, superficial to immersed, effuse, brownish, comprising branched, septate, thick-walled hyphae. Conidiophores 3–6.5 μm ($\overline{\mathrm{x}}$ = 6.5 μm, n = 20) high, 1–3 μm ($\overline{\mathrm{x}}$ = 2 μm, n = 20) wide, micronematous, hyaline to brown, unbranched, straight or curved, erected from prostrate hyphae at the center of the circular colonies, thick walled, septate, smooth walled. Conidiogenous cells holoblastic, short, cylindrical, hyaline, thick walled, terminal. Conidia dimorphic; Lenticular conidia 30–35 × 20–25 µm ($\overline{\mathrm{x}}$ = 32 × 22 µm, n = 20), turbinate, subglobose to ellipsoidal, comprising dark-brown cells in the center, subhyaline to pale brown in peripheral cells, in lateral view obovoid, dark-brown at the center, whitish to pale brown at lower and upper ends, sometimes carrying remnants of conidiogenous cells at base; Cylindrical conidia 30–35 × 15–21 µm ($\overline{\mathrm{x}}$ = 32 × 18 µm, n = 20), with 2 rows of 3–4 cells in each column, broad at the lower cells, granulate, with lower cells usually hyaline, cylindrical, constricted at the septum, turbinate and dark brown at upper cells.

Culture characteristics: Conidia germinated within 8–12 h in PDA; colonies grew rapidly reaching around 40–50 mm after one month. Obverse: colonies circular, flat, radiate, coloration from hyaline turn to brown with the maturity, pale brown at the center, black-brown at the margin, clearly visible sparse mycelium extended. Reverse: deep brown in the middle area, brown near the margin, visible extended mycelium, without pigments produced in PDA.

Substratum: Phoenix rupicola [77]; Tectona grandis [78]; Decayed branch of Mangifera indica (this study).

Distribution: Chandigarh, India [77]; Chiang Rai Province, Thailand [78]; Yunnan Province, China (this study).

Material examined: China, Yunnan Province, Honghe, Menglong Village, on a dead and decaying branch of Mangifera indica (102°50′11′′ E, 23°41′01″ N, 500 m), 22 December 2020, E.F. Yang, HHE003 (HKAS 122676), living culture, KUMCC 21-0453. GenBank numbers: KUMCC 21-0453 = ITS: OQ379016, LSU: OQ379427, SSU: OQ372924, tef1-α: OQ379001, rpb2: OQ378994.

Notes: Our isolate largely matches Hermatomyces indicus (holotype: PAN 30900) based on morphological characteristics. Our strain and Hermatomyces indicus were comparable by two types of conidia. The lenticular conidia subglobose to ellipsoidal, with dark brown cells in the center, surrounded by pale peripheral cells at the periphery, with size (30–35 × 20–25 µm vs. 31 × 21.8 µm). Cylindrical conidia hyaline, with 2 rows of 3–4 cells in each column, brown at apex, becoming hyaline towards the lower side, constricted at the septum, containing numerous oil globules, with size (30–35 × 15–25 µm vs. 22–36 × 11–22 µm) [77]. Moreover, the BLASTn search results of ITS, LSU, tef1-α and rpb2 showed our isolate (KUMCC 21-0453) is highly similar to Hermatomyces indicus strains (MFLUCC 14-1143, 14-1144, 14-1145) (>99%). The multi-gene phylogenetic trees indicated that our isolate clusters well with Hermatomyces indicus strains with high statistical support (Figure 15). Therefore, we report our isolate Hermatomyces indicus (KUMCC 21-0453) as a new host and geographic record from a decaying mango branch in China.

4. Discussion

To date, the family Plectosphaerellaceae includes more than 150 species in 24 genera following the publication “Outline of Fungi and fungus-like taxa—2021” [38]. Our isolate HKAS 122669 is relatively close to Acremoniisimulans (Acremoniisimulans cocois and A. thailandensis) with regard to the phylogenetic tree and sexual morphological characteristics [36,37]. In addition, BLASTn searches of SSU and LSU showed 99–100% similarity to Acremoniisimulans species; however, the BLASTn search results of ITS, tef-α and rpb2 show extremely low similarity (=<91%) with Acremoniisimulans and other genera in the Plectosphaerellaceae. Therefore, the novel species Acremoniisimulans honghensis is established based on the differences in morphological features, phylogenetic trees and molecular data. The family Tubeufiaceae contains over 400 species in 47 genera [38]. The asexual morphology of Excipulariopsis narsapurensis was described by Spooner & Kirk and Pem et al. [39,40]. However, the precise phylogenetic position of Excipulariopsis has not yet been determined due to the absence of molecular data. Our study is the first to have solved this problem and uploaded the multiple genes to NCBI’s GenBank nucleotide database. The asexual morphologies of the genus Delonicicola are introduced herein for the first time. That will certainly help in updating our understanding of the morphology of both sexual and asexual characteristics of this taxon.

Taxonomic studies on saprobic or endophytic fungi associated with mango are still poorly conducted since most investigations have concentrated on pathogenic fungi associated with mango. However, the life modes and growth of fungi depend on a number of biotic (insect, bacterial and plant) and abiotic (habitat) factors [79,80,81,82]. Very few studies have reported saprobic fungi as a practically valuable group. For example, Botrel et al. [83] reported that saprobic Phialomyces macrosporus has the potential to be employed in the management of coffee halo blight, which caused by Pseudomonas syringae pv. garcae, Monkai et al. [84] isolated four Cytospora species from decaying leaves, and three of them (Cytospora shoreae, C. phitsanulokensis and C. chiangmaiensis) showed 60–75% growth inhibition against pathogens Colletotrichum viniferum and Fusarium sambucinum in vitro. Therefore, deep studies of saprobic fungi are necessary, especially secondary metabolites of saprobic fungi and their life mode changes. During our investigations of microfungi associated with mango in Yunnan Province (China), we observed a pattern of high fungal diversity in the mango ([16,17], in this study). Of the 28 species of fungi introduced, based on morphology and phylogeny, most belong to the order Pleosporales (11 species, 39%), with four species belonging to the order Xylariales and three species to the order Botryosphaeriales. Together with two genera (Mangifericola and Mangifericomes) and 11 new species so far established, there is certainly an incredible diversity of new species—39% (

Table 2. List of saprobic fungi associated with mango from Honghe and Baoshan, Yunnan, China (Yang et al. [16,17] and this study); the bold text indicates new species.

Class	Order	Family	Species	Herbarium No.
Dothideomycetes	Botryosphaeriales	Aplosporellaceae	Aplosporellaartocarpi	HKAS 122656
		Botryosphaeriaceae	Lasiodiplodia theobromae	HKAS 122660
		Botryosphaeriaceae	Lasiodiplodiapseudotheobromae	HKAS 122658
	Monoblastiales	Monoblastiaceae	Heleiosabarbatula	HKAS 122668
	Pleosporales	Hermatomycetaceae	Hermatomyces indicus	HKAS 122676
		Incertaesedis	Crassipariesquadrisporus	HKAS 122192
		Incertaesedis	Mangifericomes hongheensis	HKAS 122188
		Massarinaceae	Vaginatisporaamygdali	HKAS 122195
		Melanommataceae	Byssosphaeriasiamensis	HKAS 122197
		Neomassariaceae	Neomassaria hongheensis	HKAS 122191
		Paradictyoarthriniaceae	Paradictyoarthriniumdiffractum	HKAS 122194
		Parabambusicolaceae	Paramonodictys hongheensis	HKAS 122190
		Parabambusicolaceae	Paramonodictys yunnanensis	HKAS 122189
		Phaeoseptaceae	Phaeoseptummali	HKAS 122193
		Torulaceae	Torulafici	HKAS 122196
	Tubeufiales	Tubeufiaceae	Excipulariopsisnarsapurensis	HKAS 122680
Eurotiomycete	Chaetothyriales	Cyphellophoraceae	Cyphellophora hongheensis	HKAS 122661
	Mycocaliciales	Mycocaliciaceae	Chaenothecopsis hongheensis	HKAS 122672
Sordariomycetes	Calosphaeriales	Pleurostomataceae	Pleurostomaootheca	HKAS 122679
	Coronophorales	Nitschkiaceae	Fracchiaeamyricoides	HKAS 122671
	Delonicicolales	Delonicicolaceae	Delonicicolasiamense	HKAS 122662
	Diaporthales	Diaporthaceae	Diaporthe hongheensis	HKAS 122657
	Glomerellales	Plectosphaerellaceae	Acremoniisimulans hongheensis	HKAS 122669
	Sordariales	Helminthosphaeriaceae	Hilberina hongheensis	HKAS 122677
	Xylariales	Diatrypaceae	Mangifericola hongheensis	HKAS 122665
		Diatrypaceae	Paraeutypellacitricola	HKAS 122667
		Hypoxylaceae	Hypoxylon hongheensis	HKAS 122663
		Hypoxylaceae	Hypomontagnellamonticulosa	HKAS 122664

). Among these, the species from the Botryosphaeriales, Pleosporales and Xylariales are dominant saprobic fungi on mango substrates. The results of this study also suggest that numerous additional new and interesting species of fungi are likely to be discovered in the future on mango substrates.