Fresh fungal materials were collected from dead woody twigs from Honghe, Kunming, Mengla, Shangri-La and Yulong Counties, all within Yunnan Province, China, during the dry season (January, March, April) and wet season (August, September). To preserve their integrity, the specimens were transported to the laboratory in Zip lock plastic bags during the dry season and in paper bags during the wet season.

The morphology of external and internal macro-/micro-structures were observed as described in Wanasinghe et al. (2017, 2018a, 2020). Hand sections of the ascomata were mounted in distilled water and the following characteristics were evaluated and measured: ascomata diameter, height, color and shape; width of peridium; and height and diameter of ostioles. Length and width (at the widest point) of asci and ascospores. Images were captured with a Canon EOS 600D digital camera fitted to a Nikon ECLIPSE Ni compound microscope. Macroscopic images of colonies were documented using an iPhone XS Max (Apple Inc., Cupertino, CA, USA) with daylight. Measurements were made with the Tarosoft (R) Image Frame Work program, and images used for figures were processed with Adobe Photoshop CS5 Extended version 10.0 software (Adobe Systems, San José, CA, USA).

Single spore isolation was conducted by following the methods described in Wanasinghe et al. (2018b). Germinated spores were individually transferred to potato dextrose agar (PDA: 39 g/L distilled water, Difco potato dextrose) plates and grown at 20 °C in the daylight.

The living cultures were deposited at the Kunming Institute of Botany Culture Collection (KUNCC), Kunming, China. Dry herbarium materials were deposited in the herbarium of Cryptogams Kunming Institute of Botany, Academia Sinica (KUN-HKAS). MycoBank numbers have been obtained as outlined in MycoBank (http://www.MycoBank.org accessed on 21 September 2023) for the novel taxa.

Genomic DNA was extracted from the axenic mycelium as described by Phookamsak et al. (2017). Mycelia for DNA extraction from each isolate were grown on PDA for 3–4 weeks at 20 °C and total genomic DNA was extracted from approximately 150 ± 50 mg axenic mycelium scraped from the edges of the growing culture. Mycelium was ground to a fine powder with liquid nitrogen and DNA extracted using the Biospin Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux, P.R. China) following the instructions of the manufacturer. When fungi failed to grow in culture, DNA extraction was carried out directly from fruiting bodies, adhering to the protocol outlined by Wanasinghe et al. (2018b). DNA to be used as templates for Polymerase Chain Reaction (PCR) were stored at 4 °C for use in regular work and duplicated at -20 °C for long-term storage.

We used primers ITS5/ITS4 (White et al. 1990), LR0R/LR5 (Vilgalys and Hester 1990; Rehner and Samuels 1994), NS1/NS4 (White et al. 1990), EF1-983F/EF1-2218R (Liu et al. 1999; Rehner and Buckley 2005), and fRPB2-5f/fRPB2-7cR (Sung et al. 2007) to amplify sequence data for a total of five markers: the internal transcribed spacers (ITS), partial 28S large subunit rDNA (LSU), partial 18S small subunit rDNA (SSU), translation elongation factor 1-α (tef1-α), and RNA polymerase II second largest subunit (rpb2). PCR amplifications were performed following the methods described in Wanasinghe et al. (2021). We sequenced complementary strands with the same primers used for PCR amplifications and sequencing was done from a commercial sequencing provider (BGI, Ltd Shenzhen, P.R. China). The nucleotide sequence data obtained were deposited in GenBank (Table 2).

Sequences generated from different primers of the five genes were analysed with other sequences retrieved from GenBank (Table 2). Sequences with high similarity indices were determined from a BLAST search to find the closest matches with taxa in Didymosphaeriaceae, using recently published data (Du et al. 2021; Ren et al. 2022a; Sun et al. 2023). The multiple alignments of all consensus sequences, as well as the reference sequences were automatically generated with MAFFT v. 7 (Katoh et al. 2019), and manually corrected where necessary using BioEdit v. 7.0.5.2 (Hall 1999).

The single-locus datasets were examined for topological incongruence among loci for members of the analyses. The alignments were concatenated into a multi-locus alignment that was analyzed with maximum likelihood (ML) and Bayesian (BI) phylogenetic methods in the CIPRES Science Gateway (Miller et al. 2010). ML tree was obtained using RAxML-HPC2 on XSEDE v. 8.2.10 (Stamatakis 2014) with applying GTR+G+I model. Support values were obtained with 1,000 bp replicates (Felsenstein 1985). ML bootstrap values equal or greater than 75% are given above each node. The best-fit model was selected with respect to Bayesian Information Criterion (BIC) scores using the IQ-TREE web application at http://iqtree.cibiv.univie.ac.at (Trifinopoulos et al. 2016). For model selection, we restricted the pool of available models to JC, F81, HKY, SYM and GTR (Ronquist et al. 2011). BI were performed with two parallel runs of 2 M generations, using four chains in each, and retaining one tree every 100 generations. The dataset was partitioned by gene region, and a GTR + G + I model was applied to each partition, ending the run automatically when standard deviation of split frequencies dropped below 0.01 with a burn-in fraction of 0.25. A fifty percent majority rule consensus tree was obtained after discarding the first 25% of trees, and posterior probabilities were used as a measure of nodal support. The posterior probability in BI (BYPP) greater than 0.95 are given above each node. Phylograms were visualized with FigTree v1.4.0 program (Rambaut 2012) and reorganized in Microsoft power point (2019).

In our initial approach, we obtained detailed geographical distribution information for the Montagnula genus. This data was extracted from the GlobalFungi database (https://globalfungi.com, accessed on 04 December 2023), as outlined by Větrovský et al. (2020). The database provided information on the countries and precise geographical coordinates of recorded Montagnula occurrences. To visualize these occurrences, we employed a range of packages in R version 4.2.1 (R Core Team 2022), including ‘sf’ (Pebesma and Bivand 2023), ‘raster’ (Hijmans 2023), ‘rgdal’ (Bivand et al. 2022), and ‘ggplot2’ (Wickham 2011). In our map, each marker signifies an individual occurrence of Montagnula. These occurrences are visually distinguished by a color scheme, with each color denoting the specific biome from which the samples were collected, as illustrated in Fig. 2a. Additionally, we have developed two donut charts, showcased in Fig. 2b, c, which effectively illustrate the distribution of Montagnula sequences. These charts present the sequence abundance as a percentage of the total, segmented across various biomes and continents, providing a clear visual breakdown of their distribution. Furthermore, we have gathered Environmental DNA (eDNA) data from diverse sources in metabarcoding studies focusing on fungi, as found in the GlobalFungi database (Fig. 3). This dataset included specifics about eDNA sources, locations of the studies, and the sequence abundance of Montagnula sequences. It is important to note that the sequence abundance in metabarcoding studies might not always accurately represent the actual abundance of species in a habitat. Nonetheless, these data can provide valuable insights into the potential rarity or prevalence of the group in the eDNA source. We analyzed the sequence abundance in diverse eDNA samples from different continents. Before visualization, the abundance values were normalized via a logarithmic transformation to ensure a standardized and comparable presentation of Montagnula sequence abundance. Post-transformation abundance data were visualized using the ‘ggplot2’ package, aiding in highlighting the focus areas of metabarcoding and identifying the environmental sample types from which Montagnula sequences were derived across various continents (Figs 2, 3).

To illustrate the host specificity of Montagnula species, we utilized detailed information regarding host species from the literature (Table 1). This enabled us to create informative bar plots displaying the host preferences of Montagnula species (Fig. 4). This information was visualized using the ‘ggplot2’ package in R.

In order to examine the evolutionary relationships of our new strains within Montagnula, phylogenetic analyses were performed based on the combined SSU, LSU, ITS, tef1-α, and rpb2 DNA sequences of 56 representatives of the genus and two strains from Neokalmusia jonahhulmei (KUMCC 21-0818, KUMCC 21-0819) as the outgroup taxon. The full dataset consisted of 4,268 characters including gaps (18S = 1,023 characters, 28S = 896, ITS = 508, tef1-α = 885, rpb2 = 956). The RAxML analysis of the combined dataset yielded a best-scoring tree with a final ML optimization likelihood value of -14,343.052271. The matrix had 1004 distinct alignment patterns, with 23.88% undetermined characters or gaps. Parameters for the GTR + I + G model of the combined amplicons were as follows: Estimated base frequencies; A = 0.244145, C = 0.256118, G = 0.269851, T = 0.229886; substitution rates AC = 1.815063, AG = 3.954334, AT = 1.414215, CG = 1.362941, CT = 10.779403, GT = 1.000; proportion of invariable sites I = 0.559204; and gamma distribution shape parameter α = 0.542439. The Bayesian analysis ran 1,675,000 generations before the average standard deviation for split frequencies reached below 0.01 (0.009994). The analyses generated 16,751 trees, from which we sampled 12,564 trees after discarding the first 25% as burn-in. The alignment contained a total of 1,005 unique site patterns. The BI and ML trees were not in conflict; the ML tree is shown in Fig. 1. Where applicable, the phylogenetic results obtained (Fig. 1) are discussed in the descriptive notes below.

Figure 1. 

Phylogenetic analysis of SSU, LSU, ITS, tef1-α, and rpb2 of the Montagnula. Species names given in bold are ex-type, ex-epitype and ex-paratype strains. Species names highlighted in blue are generated from this study. Branch support of nodes ≥75% ML BS and ≥0.95 PP is indicated above the branches. The genus Montagnula is depicted within a pale gray box, with new species highlighted in white, and the outgroup indicated by a blue box.

Figure 2. 

Geographical distribution of Montagnula species with known ITS sequence data. a the map summarizes data from the GlobalFungi database (shown by circles). Each circle symbolizes a unique sample, with each color representing the specific biome from which it has been collected b the distribution of Montagnula sequences as a percentage of total abundance across different biomes c the distribution of Montagnula sequences as a percentage of total abundance across different continents. See Suppl. material 1 for primary data.

We conducted a thorough study of a compilation of data derived from multiple metabarcoding studies, which documented the occurrence of Montagnula species worldwide, excluding Antarctica. Among the continents, the highest number of studies were recorded in Asia, Australia, Europe, and North America (Fig. 2). These studies encompassed a diverse range of 11 distinct sources, revealing that sediments and “other” sources yielded the highest number of sequences (Fig. 3). Across different continents, the sequences obtained from various sources exhibited moderate similarity. However, in regions such as Asia, Australia, Europe, and North America, studies revealed Montagnula species from a diverse array of sources, in contrast to other studies, which identified species from a more limited selection of sources. Furthermore, in culture-based investigations, the primary focus was on extracting Montagnula species from plant substrates originating from 45 distinct plant families (Fig. 4). Among these families, Poaceae yielded the most substantial number of isolated species, followed by Asparagaceae and Fabaceae. Additionally, two records were also detected in mushrooms and human skin samples.

Figure 3. 

The distribution of Montagnula occurrences across oceans, continents and various substrates, as documented in the existing literature. On the x-axis, the logarithmic abundance of each record for different sources is displayed.

Figure 4. 

The species richness of recorded Montagnula species across different plant families (Table 1).

Pleosporales Luttr. ex M.E. Barr, Prodromus to class Loculoascomycetes: 67 (1987)

Didymosphaeriaceae Munk, Dansk botanisk Arkiv 15 (2): 128 (1953)

 Montagnula aquilariae T.Y. Du & Tibpromma, Mycosphere 14 (1): 705 (2023)

MycoBank No: 846332

Fig. 5

Description

Saprobic on dead woody litter of an unknown deciduous host. Teleomorph Ascomata 450–600 μm high × 480–550 μm diam., immersed to semi-erumpent, gregarious or rarely clustered, globose to subglobose, ostiolate. Ostiole 120–220 × 70–110 µm (x– = 139 × 89 μm, n = 5), papillate, central, straight, dark brown to black, filled with hyaline cells, periphyses are lacking. Peridium 20–40 μm thick on the sides and can reach up to 60 μm near the apex, with an outer layer consisting of heavily pigmented cells that have thick walls and exhibit a textura angularis to textura globulosa texture at the apex, textura angularis texture at the sides and base; the innermost layer consists of narrow, hyaline compressed rows of cells that merge with pseudoparaphyses. Hamathecium of 2–4 μm broad, dense, narrow, branched, cellular pseudoparaphyses. Asci 100–120 × 16–22 µm (x– = 110.8 × 18.4 μm, n = 20), bitunicate, fissitunicate, cylindrical-clavate to clavate, pedicel 30–50 μm long, 8-spored, biseriate, with a minute ocular chamber best seen in immature ascus. Ascospores 20–25 × 8.5–11 µm (x– = 21.8 × 9.6 μm, n = 30), ellipsoidal to narrowly oblong, straight or somewhat curved, ends conically rounded, golden-brown to dark brown, 1-septate, constricted at the septum, large guttules in each cell, verruculose, with a thin mucilaginous sheath. Anamorph Undetermined.

Habitat and distribution

This species is found in terrestrial habitats of Yunnan, China, specifically inhabiting dead woody twigs of deciduous hosts, including Aquilaria sinensis (Hyde et al. 2023, this study).

Material examined

China, Yunnan Province, Kunming City, Kunming Institute of Botany (25.141723°N, 102.750013°E, 1970 m), on dead woody litter of an unidentified plant, 24 April 2022, L. Qinxian, KIB22-17-1 (HKAS 126542), living culture KUNCC 23-14430; ibid. 25.141487°N, 102.748863°E, 1982 m, K2B22-17-3 (HKAS 126543), living culture KUNCC 23-14431; ibid. K2B22-17-4 (HKAS 126544), living culture KUNCC 23-14432.

Notes

Montagnula aquilariae was recently introduced by Hyde et al. (2023) based on samples obtained from Aquilaria sinensis in Xishuangbanna, Yunnan Province. In our new collections, three strains (KUNCC 23-14430, KUNCC 23-14431, KUNCC 23-14432) exhibited a monophyletic relationship with the previously known strains of Montagnula aquilariae (KUNCC 22-10815 [ex-type] and KUNCC 22-10816). Through further morphological, ecological, and nucleotide (SSU, LSU, ITS, tef1-α) comparisons, we have confirmed that these new strains indeed belong to Montagnula aquilariae. Our research also provides additional insights into the characteristics of Montagnula aquilariae. Specifically, we report the verruculose feature of the ascospores and present rpb2 sequence data for this fungus, advancing our knowledge of its morphological and genetic attributes.

Figure 5. 

Montagnula aquilariae (HKAS 126542) a, b ascomata on natural wood surface c vertical section through an ascoma d ostiolar neck e peridium cells at the apex f peridium cells at the side g pseudoparaphyses h–l asci m–r ascospores (see verruculose feature of the ascospore in r) s, t culture characters on PDA (s = above, t = reverse). Scale bars: 100 μm (c, d); 50 μm (e); 10 μm (e–g, m–r); 20 μm (h–l).

 Montagnula lijiangensis Wanas., sp. nov.

MycoBank No: 850093

Fig. 6

Etymology

The specific epithet “lijiangensis” refers to Lijiang, Yunnan Province, where the holotype was collected.

Holotype

HKAS 126541.

Description

Saprobic on dead woody litter of Quercus sp. Teleomorph Ascomata 500–700 μm high × 500–600 μm diam., immersed, gregarious or rarely clustered, globose to subglobose, ostiolate. Ostiole 100–140 × 80–120 µm (x– = 125 × 96 μm, n = 5), apapillate, central, straight, filled with hyaline cells. Peridium 20–30 μm thin on the sides and can reach up to 70 μm near the apex, with an outer layer consisting of heavily pigmented cells that have thick walls and exhibit a textura angularis texture at the apex, textura angularis texture at the sides and base; the innermost layer consists of narrow, hyaline compressed rows of cells. Hamathecium of 3–7.5 μm broad, dense, narrow, branched, cellular pseudoparaphyses that are swollen at the base. Asci 130–160 × 20–26 µm (x– = 152.8 × 23.9 μm, n = 20), bitunicate, fissitunicate, cylindrical-clavate to clavate, pedicel 30–60 μm long, 8-spored, uni to biseriate, with a minute ocular chamber best seen in immature ascus. Ascospores 22–26 × 10–14 µm (x– = 24.8 × 11.8 μm, n = 30), ellipsoidal to narrowly oblong, mostly straight, with conically rounded ends at the immature stage that become rounded when mature, golden-brown to dark brown, 1-septate and constricted at the septum, with large guttules in each cell, verruculose, surrounded by a thick mucilaginous sheath. Anamorph Undetermined.

Figure 6. 

Montagnula lijiangensis (HKAS 126541, holotype) a, b ascomata on natural wood surface c vertical section through an ascoma d ostiolar neck and peridium cells at the apex e pseudoparaphyses f–i asci j–o ascospores (see verruculose feature of the ascospore in k). Scale bars: 100 μm (c); 20 μm (d, f–i); 10 μm (e–o).

Habitat and distribution

This species is found in terrestrial habitats of Yunnan, China, inhabiting dead woody twigs of deciduous hosts (this study).

Material examined

China, Yunnan Province, Lijiang, Yulong County (26.86389°N, 99.824738°E, 2725 m), on dead woody litter of Quercus sp. (Fagaceae), 17 August 2021, L. Qinxian, STX09-03-1 (holotype, HKAS 126541, ibid. 26.863484°N, 99.824548°E, 2706 m, STX09-03-3 (HKAS 126540).

Notes

The analysis of two newly generated sequences revealed a monophyletic clade in our phylogenetic analysis (Fig. 1), demonstrating a close phylogenetic relationship to Montagnula aquilariae. This relationship is further supported by morphological features such as asci and ascospores. However, a comparison of nucleotide differences (without gaps) between these two clades (KUNCC 22-10815 and KUNCC 23-14430 vs HKAS 126541) showed 12/508 (2.3%) differences in the ITS region, 15/885 (1.7%) differences in the tef1-α region, and 19/956 (2%) differences in the rpb2 region.

 Montagnula menglaensis Wanas., sp. nov.

MycoBank No: 850094

Fig. 7

Etymology

The specific epithet “menglaensis” refers to Mengla County, Yunnan Province, where the holotype was collected.

Holotype

HKAS 130318.

Description

Saprobic on dead culms of Indocalamus tessellatus (Munro) Keng f. Teleomorph Ascomata 200–300 μm high × 240–320 μm diam., immersed, gregarious or rarely clustered, globose to subglobose. Peridium 10–25 μm thin with an outer layer consisting of heavily pigmented cells that have thick walls and exhibit a textura angularis texture at the sides and base; the innermost layer consists of narrow, hyaline compressed rows of cells. Hamathecium of 3–7.5 μm broad, dense, branched, cellular pseudoparaphyses that are swollen at some septa. Asci 60–80 × 9–11 µm (x– = 71 × 9.8 μm, n = 15), bitunicate, fissitunicate, cylindrical-clavate, pedicel 15–30 μm long, 8-spored, uni to biseriate, with a minute ocular chamber best seen in immature ascus. Ascospores 10.5–14 × 4.5–5.5 µm (x– = 12.6 × 5.1 μm, n = 20), ellipsoidal, mostly straight, with conically rounded ends, golden-brown to dark brown, 1-septate and constricted at the septum, upper cell wider than the lower cell, with large guttules in each cell, verruculose, and surrounded by a thin mucilaginous sheath which is thicker at both ends. Anamorph Coelomycetous on PDA. Conidiomata pycnidial, gregarious, immersed to superficial, globose to subglobose, dark brown to black. Pycnidial wall thin, composed of brown cells of textura angularis. Conidiogenous cells did not observed. Conidia 2.3–3.3 × 1.4–2 μm (x– = 3 × 1.7 μm, n = 30), hyaline, aseptate, round to oblong or ellipsoidal, with small guttules.

Figure 7. 

Montagnula menglaensis (HKAS 130318, holotype) a–c ascomata on natural wood surface d, e vertical section through ascomata f, g pseudoparaphyses h peridium i–k asci l, m ascospores (see verruculose feature of the ascospore in n) o, p culture characters on PDA (o = above, p = reverse) q, r conidiomata s pycnidial wall t conidia. Scale bars: 100 μm (d, e); 10 μm (f–h, l–n, s, t); 20 μm (i–k).

Culture characteristics

Ascospores germinated on PDA within 24 h. Following a two-week incubation period at 25 °C, the colonies on PDA medium reached a diameter of 5 cm. These colonies exhibited an undulate margin, initially appearing creamy whitish and transitioning to orange, raised in the center. The colonies were orange at the center and a creamy orange towards the periphery when observed from the reverse side.

Habitat and distribution

This species is found in terrestrial habitats of Yunnan, China, inhabiting dead woody twigs of deciduous hosts (this study).

Material examined

China, Yunnan Province, Xishuangbanna, Mengla County (21.588394°N, 101.435042°E, 776 m), on dead culms of Indocalamus tessellatus, 29 January 2022, L. Qinxian, ML23-7-3 (holotype, HKAS 130318), ex-type KUNCC 23-14424; ibid. 21.589178°N, 101.435752°E, 782 m, ML23-7-2 (HKAS 130316), living culture KUNCC 23-14422; ibid. ML23-7-5 HKAS 130317), living culture KUNCC 23-14423.

Notes

Montagnula menglaensis is described as a novel species based on its holomorph. The anamorph of Montagnula is rarely encountered; however, Crous et al. (2020) recently reported Montagnula cylindrospora based on its anamorphic features. The conidia of Montagnula menglaensis resemble to those of M. cylindrospora, although the latter fungus exhibits a more cylindrical shape.

 Montagnula shangrilana Wanas., sp. nov.

MycoBank No: 850095

Fig. 8

Etymology

The specific epithet “shangrilana” refers to Shangri-La, Yunnan Province, where the holotype was collected.

Holotype

HKAS 126539.

Description

Saprobic on dead woody litter of Rhododendron sp. Teleomorph Ascomata 120–180 μm high × 150–210 μm diam., immersed to semi-erumpent, gregarious or rarely clustered, globose to subglobose, ostiolate. Ostiole 80–110 × 50–80 µm (x– = 100 × 64 μm, n = 6), papillate, central, straight, filled with hyaline cells. Peridium 10–20 μm thin on the sides and can reach up to 40 μm near the apex, with an outer layer consisting of heavily pigmented cells that have thick walls and exhibit a textura angularis arrangement at the apex, textura angularis texture at the sides; the innermost layer consists of hyaline compressed rows of cells. Hamathecium of 2–4.5 μm broad, dense, branched, cellular pseudoparaphyses. Asci 90–140 × 20–30 µm (x– = 116.2 × 24 μm, n = 10), bitunicate, fissitunicate, cylindrical-clavate, pedicel 25–40 μm long, 8-spored, uni to biseriate, with a minute ocular chamber best seen in immature ascus. Ascospores 48–60 × 17–22 µm (x– = 55.8 × 19.3 μm, n = 20), ellipsoidal to narrowly oblong, mostly straight, with conically rounded ends at the immature stage that become rounded when mature, golden-brown to dark brown, 3-septate, with large guttules in each cell, verruculose, surrounded by a thick mucilaginous sheath. Anamorph Undetermined.

Figure 8. 

Montagnula shangrilana (HKAS 126541, holotype) a ascomata on natural wood surface b vertical section through an ascoma c pseudoparaphyses d peridium cells e–h asci i–o ascospores (see verruculose feature of the ascospore in o). Scale bars: 100 μm (b); 10 μm (c, d, j–o); 20 μm (e–h).

Culture characteristics

Ascospores germinated on PDA within 24 h. Following a two-week incubation period at 25 °C, the colonies on PDA medium reached a diameter of 5 cm. These colonies exhibited a filiform margin, initially appearing whitish and transitioning to greenish gray, raised in the center. The colonies were grey at the center and a greenish gray towards the periphery and radiated when observed from the reverse side.

Habitat and distribution

This species is found in terrestrial habitats of Yunnan, China, inhabiting dead woody twigs of deciduous hosts, in a subalpine environment (this study).

Material examined

China, Yunnan Province, Diqing Tibetan Autonomous Prefecture, Shangri-La (27.289707°N, 100.034477°E, 2744 m), on dead woody litter of Rhododendron sp. (Ericaceae), 22 August 2021, L. Qinxian, WTS8-2-2 (holotype, HKAS 126539), ex-type KUNCC 23-14434; ibid. (27.290007°N, 100.035233°E, 2833 m, WTS8-2 (HKAS 126538), living culture KUNCC 23-14433.

Notes

In the combined SSU, LSU, ITS, tef1-α, and rpb2 phylogenetic analysis, two strains of Montagnula shangrilana (HKAS 126538, HKAS 126539) formed a monophyletic clade closely related to M. camporesii (MFLUCC 16-1369), M. cirsii (MFLUCC 13-0680), and M. scabiosae (MFLUCC 14-0954). While there were slight variations in size, shape, and color, all four species shared the common characteristic of 3-transversely septate ascospores. The sequence data of Montagnula camporesii, M. cirsii, and M. scabiosae showed no significant differences in their base pair comparisons, suggesting that they may be conspecific. Morphologically, these three species exhibited clavate asci and ellipsoid to fusiform, brown, overlapping, 3-septate ascospores. In contrast, our newly discovered species differed from these three species by 10/508 (1.96%) differences in the ITS region, 13/885 (1.5%) differences in the tef1-α region, and 15/956 (1.56%) differences in the rpb2 region (only M. camporesii possesses rpb2).

 Montagnula thevetiae Wanas., sp. nov.

MycoBank No: 850096

Fig. 9

Etymology

The specific epithet “thevetiae” refers to the host Thevetia peruviana from which the holotype was isolated.

Holotype

HKAS 126964.

Description

Saprobic on dead twigs of Thevetia peruviana. Teleomorph Ascomata 140–160 μm high × 150–190 μm diam., immersed, gregarious or rarely clustered, globose to subglobose, ostiolate. Ostiole 40–65 × 50–90 µm (x– = 50 × 78 μm, n = 6), papillate, central, straight, filled with hyaline to brown cells. Peridium 10–20 μm thin on the sides and can reach up to 30 μm near the apex, with an outer layer consisting of heavily pigmented cells that have thick walls and textura angularis arrangement, the inner layer consists of hyaline compressed rows of cells. Hamathecium of 2–3.5 μm broad, dense, branched, cellular pseudoparaphyses. Asci 110–160 × 25–35 µm (x– = 126.4 × 30.3 μm, n = 12), bitunicate, fissitunicate, cylindrical-clavate, pedicel 25–35 μm long, 8-spored, uni to biseriate, with a minute ocular chamber best seen in immature ascus. Ascospores 30–40 × 11.5–14 µm (x– = 37.3 × 12.8 μm, n = 20), ellipsoidal to narrowly oblong, straight to curved, with conically rounded ends, brown to dark brown, 1-septate, constricted at the septum, with large guttules in each cell, verruculose, surrounded by a thin mucilaginous sheath. Anamorph Undetermined.

Figure 9. 

Montagnula thevetiae (HKAS 126564, holotype). a, b ascomata on natural wood surface c vertical section through an ascoma d closeup of ostiole e pseudoparaphyses f–h asci j–l ascospores m, n culture characteristics on PDA (m = above, n = reverse). Scale bars: 100 μm (c); 50 μm (d, f–h); 10 μm (e, i–l).

Culture characteristics

Ascospores germinated on PDA within 24 h. Following a two-week incubation period at 25 °C, the colonies on PDA medium reached a diameter of 4 cm. These colonies exhibit an irregular, flattened to slightly raised morphology and display various color sectors ranging from white, creamy orange to pale brown. The reverse side of the colonies appears creamy orange, with occasional dark patches that can be observed.

Habitat and distribution

This species is found in terrestrial habitats of Yunnan, China, inhabiting dead woody twigs of Thevetia peruviana (this study).

Material examined

China, Yunnan Province, Kunming city, Kunming Institute of Botany (25.142238°N, 102.750354°E, 1971 m), on dead twigs of Thevetia peruviana, 24 April 2022, L. Qinxian, K2B22-26-2 (holotype, HKAS 126964), ibid. (25.140859°N, 102.749045°E, 1968 m, K2B22-26 (HKAS 126963).

Notes

Montagnula thevetiae is isolated from the dead twigs of Thevetia peruviana. The newly obtained sequences of this fungus formed a monophyletic clade closely related to Montagnula menglaensis. Morphologically, they share similarities in having 1-septate ascospores, although Montagnula thevetiae exhibits a darker pigmentation. On the other hand, Montagnula thevetiae differs from M. menglaensis by 15/1023 (1.46%) differences in the SSU region, 19/895 (2.12%) differences in the LSU region, 32/508 (6.3%) differences in the ITS region, 27/885 (3%) differences in the tef1-α region, and 86/956 (9%) differences in the rpb2 region.

The study of lignicolous microfungi in Yunnan Province resulted in the collection of eight Montagnula species, including four novel species. This study contributes to our understanding of the diversity and distribution of Montagnula species and provides insight into the ecological roles played by these fungi in their respective habitats. Montagnula aquatica was previously documented as occurring on submerged decaying wood within a freshwater habitat in Thailand (Sun et al. 2023). However, our recent collection of this species was obtained from a terrestrial habitat in China. The holotype was collected in the Bandu District of the Chiang Rai Province, situated at an approximate elevation of 400–450 m and characterized by a tropical climate. The collection site was near to a waterfall (Sun et al. 2023). In contrast, our new collections were made in the Honghe region of Yunnan Province, which possesses an elevation of approximately 1200 m. The local environment in this region is characterized by poor, eroded soils, steep valleys, and a subtropical climate. This observation suggests that Montagnula aquatica may possess an adaptable nature, enabling it to thrive in a wide range of habitats across diverse geographic locations. Montagnula aquilariae, another species within the genus, has been identified in the terrestrial habitats of Yunnan, China. It specifically colonizes dead woody twigs of deciduous hosts, including Aquilaria sinensis (Hyde et al. 2023). The holotype of this species was collected from a hilly area in Nanmo, Menghai and Xishuangbanna, situated at an elevation of ~1100 m and characterized by a tropical climate. Additional collections were made from Kunming, located within the same province but at an elevation of ~2000 m, and characterized by a warm and temperate climate. Montagnula chromolaenicola has been observed in terrestrial habitats in Thailand, particularly on dead stems of Chromolaena odorata (Mapook et al. 2020). The holotype of this species was collected from the Mae Yen mountainous area of Mae Hong Son Province, at an elevation of ~900 m. The local environment of this area exhibits a tropical savanna climate. In our study, we collected this fungus from a terrestrial habitat within the steep valleys of subtropical Honghe, Yunnan, China. In this region, Montagnula chromolaenicola was found to inhabit the dead woody litter of deciduous hosts. Montagnula donacina has been reported across various terrestrial habitats worldwide, with the majority of records originating from India (Table 1). This species primarily associates with hosts from the Poaceae family. In our study, we collected Montagnula donacina from the subtropical Honghe region in China, specifically on decaying woody litter at an elevation of ~600 m. Montagnula lijiangensis was collected from terrestrial habitats at a high elevation of ~2725 m. This species was found on dead woody litter of Quercus sp. within an environment characterized by a mild subtropical highland climate. Montagnula menglaensis was discovered in the terrestrial habitats of Mengla County, Yunnan, China. It was observed colonizing dead culms of Indocalamus tessellatus. The local environment of this region exhibits a tropical savanna climate, with an elevation of ~800 m. Montagnula shangrilana was found in the terrestrial habitats of Shangri-La, Yunnan, China, where it inhabits dead woody twigs of Rhododendron sp. This species has also been observed at higher elevations, reaching ~2800 m, within an environment characterized by a humid continental climate. Montagnula thevetiae was discovered within the terrestrial habitat of the botanical garden at the Kunming Institute of Botany in Yunnan, China. This species was found colonizing dead woody twigs of Thevetia peruviana. The collection site is situated at an elevation of ~2000 m and experiences a warm and temperate climate.

In a recent study conducted by Sun et al. (2023), Montagnula chromolaenicola, M. puerensis, M. saikhuensis, and M. thailandica were regarded as the synonyms of M. donacina (Wanasinghe et al. 2016). This decision was based on the absence of clear branches in their phylogenetic tree and the close morphological resemblance between these species. However, upon further examination, it was observed in this study that only Montagnula donacina and M. thailandica appear to be conspecific, based on combined gene analyses (Fig. 1). When informative sequence data such as tef1-α or rpb2 were added to the analysis for Montagnula chromolaenicola, M. puerensis, M. saikhuensis, and M. thailandica, distinct branches and independent lineages were observed (Fig. 1). This suggests that these species are separate entities. Notably, two sequences of M. donacina (HVVV01 and HFG07004) were found to be monophyletic with the type strain of Montagnula chromolaenicola (MFLUCC 17-1469), indicating that they belong to the latter species. In the case of Montagnula camporesii (MFLUCC 16-1369), M. cirsii (MFLUCC 13-0680), and M. scabiosae (MFLUCC 14-0954), the type strains formed a monophyletic lineage as a single species. Nucleotide base pair comparison of LSU, SSU, and ITS between these three strains did not reveal any differences. Therefore, it is suggested that Montagnula camporesii and M. cirsii should be synonymized under M. scabiosae, as it is the oldest name. However, it is important to note that this taxonomic clarification was not within the scope of our study, and future studies should compare the morphology of the holomorphs to resolve any remaining taxonomic confusion. Apart from these two clades, all other species formed distinct lineages in the multi-gene phylogenetic analysis. Out of the accepted 54 species in this genus, sequence data are currently available for only 28 species, including the four new species introduced in this study. This leaves approximately 48% of the species in need of phylogenetic sorting. Hence, future studies based on taxonomy should prioritize obtaining DNA sequence data for the remaining species. They should aim to acquire informative sequence data, such as tef1-α and rpb2, for all strains, and focus on revising the taxonomy of all species within the genus Montagnula.

The genus Montagnula exhibits rare reporting of its anamorphic features, with only one species, M. cylindrospora, described from its anamorph in addition to our study (Crous et al. 2020). This finding has helped confirm its phylogenetic placement within the genus. The teleomorph, rather than the anamorph, appears to be more commonly observed in the natural environment. The majority of Montagnula species produce immersed or semi-immersed ascomata, which are globose to subglobose in shape and possess a central papillate ostiole. However, there are a few exceptions, such as M. camporesii, M. cirsii, and M. longipes, which have been reported to have superficial ascomata. Upon closer examination, it becomes apparent that Montagnula camporesii and M. cirsii actually have semi-immersed ascomata, as illustrated in Hyde et al. (2016, 2020). It is worth mentioning that Aptroot (1995) did not illustrate the ascomata, and their orientation remains unclear. Additionally, only one species, Montagnula bellevaliae, has been reported to possess an eccentric ostiole (Hongsanan et al. 2015). The peridium cells of Montagnula species commonly exhibit a thick-walled arrangement with a textura angularis pattern. Notably, the cells near the apex are often thicker compared to those on the sides and base walls. A distinguishing characteristic for species within this genus is the presence of swollen cells in pseudoparaphyses. The asci, typically exhibit a cylindrical to clavate shape with a prominent pedicel. Ascospores in Montagnula are predominantly described as ellipsoidal to fusiform, pigmented, and septate. The majority of species (>15) have ascospores with a single septum, while some species, including M. dasylirionis, M. dura, M. infernalis, M. mohavensis, M. phragmospora, M. spinosella, and M. yuccigena, have been reported to possess muriform spores (Du et al. 2023). The remaining species have ascospores with either 3 or 5 septa. A distinct characteristic within the genus is the verruculose surface texture of the ascospores which is neglected by most of the studies. Only Montagnula appendiculata, M. chiangraiensis, and M. chromolaenae have been documented to possess polar appendages (Aptroot 2004; Mapook et al. 2020).

The information we gathered from our culture-based investigations revealed that Montagnula species were found on 105 genera in 45 distinct plant families, in 55 countries (Table 1). This highlights the wide ecological range and adaptability of Montagnula species across different hosts and geographic regions. Among the plant families, Poaceae emerged as the most significant contributor, yielding the highest number of isolated Montagnula species (Fig. 4). This finding suggests a potential association between Montagnula species and grasses, indicating the ecological importance of the Poaceae family in the life cycle and development of Montagnula species. Furthermore, Montagnula species were also detected in other plant families, such as Asparagaceae and Fabaceae, indicating their potential interactions with a diverse range of host plants. Among the more than 100 plant genera associated with Montagnula species, Agave (Asparagaceae), Opuntia (Cactaceae), Phoenix (Arecaceae), Ammophila (Poaceae), and Yucca (Asparagaceae) were found to have the greatest number of species, collectively representing 25% of the total count. This highlights the potential preference of Montagnula species for these specific plant genera within their respective families. The analysis of country-wise distribution revealed that India had the highest number of Montagnula entries (Table 1). The majority of these entries were attributed to Montagnula donacina, indicating a wide distribution of this species in India. Among the countries where Montagnula species were reported, China exhibited the highest diversity with nine different species, followed by Italy and the USA with seven different species each. This suggests regional variations in the diversity and distribution of Montagnula species. Interestingly, our study also detected Montagnula species in mushrooms and human skin samples, indicating their presence in alternative sources and potential interactions with other organisms. This highlights the need for further investigation into the ecological roles and potential impacts of Montagnula species in these non-traditional habitats. Except for Antarctica, Montagnula donacina has been reported from various countries across all six continents. Additionally, it has been identified in 25 different plant families. Investigating the reasons behind its wide distribution and adaptation to diverse ecological conditions would be intriguing. Future studies should focus on the morphological features, secondary metabolites, and gene data-based analyses of the species. To date, only six studies, including this one, have provided entries featuring both morphology and DNA-based sequence data evidence (Pitt et al. 2014; Zhao et al. 2018; Ren et al. 2022a; Li et al. 2023; Sun et al. 2023).

These findings elucidate the global distribution and ecological preferences of Montagnula species, highlighting the significance of different sources and plant families in their occurrence and potential ecological interactions. The wide range of sources from which species were identified suggests their adaptability and potential ecological roles in various ecosystems. The study also has important implications for our understanding of the ecology and biology of Montagnula fungi. All of the new species described in this study were found to be associated with dead wood, indicating the role that these fungi play in the decomposition of organic matter in forest ecosystems. We suggest that future studies could investigate the functional roles played by Montagnula fungi in ecosystem processes, such as carbon and nutrient cycling.

In addition to the taxonomic novelties, this study utilized metabarcoding data from the GlobalFungi database (Větrovský et al. 2020) to gain insights into the global diversity and distribution of Montagnula. Metabarcoding is a valuable tool that allows for the rapid identification of multiple species from complex environmental samples, providing confirmation of their presence in specific habitats. The analysis of multiple metabarcoding studies provided comprehensive information on the occurrence and distribution patterns of Montagnula species worldwide. The distribution of Montagnula across diverse biomes underscores their remarkable ecological adaptability and diversity. Forests, constituting 61% of their habitats, emerge as the predominant biome, indicating a strong preference or adaptation of the genus to forest ecosystems. Grasslands, accounting for 18%, also represent a significant habitat, suggesting the versatility in adapting to open and semi-open landscapes of them. Croplands (6%) and shrublands (7%) further exemplify the adaptability of Montagnula, thriving in both cultivated areas and natural, low-vegetation environments. Notably, woodlands and anthropogenic areas, representing 2% and 1% respectively, highlight the ability to exist in moderately wooded areas and regions significantly influenced by human activity. Additionally, their presence in aquatic environments, deserts, and wetlands, each accounting for 1% of their habitats, along with a notable 3% in mangroves, reflects the broad ecological niche of them. The marginal occurrence in tundras (0.1%) suggests a limited but notable ability to survive in extreme cold climates. The presence of Montagnula in such varied biomes underscores its ecological versatility and the importance of diverse habitats in understanding its biogeography.

The presence of Montagnula species has been documented in various regions of Africa, Arctic Ocean, Asia, Australia, Europe, Indian Ocean, North America, Pacific Ocean and South America indicating their widespread occurrence and ecological significance in these areas. In Asia, Montagnula species have been observed in multiple countries, including China, India, Indonesia, Iran, Japan, Malaysia, South Korea, Thailand and others (Suppl. material 1). The diverse range of habitats in these regions, such as freshwater habitats, terrestrial environments, and mountainous areas, offer suitable ecological niches for Montagnula colonization and growth. The detection of Montagnula species in different ecological contexts within Asia suggests their ability to adapt to various local conditions and substrates, contributing to their wide distribution across the continent. For example, in China, Montagnula species have been found in diverse habitats ranging from aquatic environments to forests and grasslands (Suppl. material 1), indicating their adaptability to different ecosystems. This adaptability may be attributed to their ability to utilize a wide range of organic materials as substrates, including decaying plant remains.

Australia also exhibits a notable presence of Montagnula species, indicating their occurrence in diverse habitats throughout the continent (Bissett et al. 2016; Luis et al. 2019; Turner et al. 2019; Gui et al. 2023). The unique ecosystems in Australia, including deserts, rainforests and grasslands, provide opportunities for Montagnula to establish themselves in different ecological niches. The metabarcoding studies were used for various biomes i.e. anthropogenic, aquatic, cropland, desert, forest, grassland, mangrove, shrubland, wetland and woodland (Fig. 3). This highlights the higher presence and distribution of Montagnula in different habitats within Australia. In Europe, Montagnula species have been recorded in several countries, including Austria, Belgium, Czech Republic (highest), Estonia, France, Germany, Italy, Netherlands Slovenia, Sweden Switzerland and Spain (Suppl. material 1). The presence of Montagnula in Europe suggests their ability to adapt to different climates and ecological conditions. This broad distribution across Europe indicates the need for further investigation into the ecological preferences and potential impacts of Montagnula species in this region. For instance, studies in Europe have identified Montagnula species in different habitats, such as anthropogenic, aquatic, cropland, desert, forest, grassland, shrubland, tundra, wetland and woodland (Suppl. material 1). Africa and North America also demonstrates a diverse distribution of Montagnula species, with the majority of records coming from the South Africa, Namibia, Botswana, Zambia, Mozambique, Kenya, Kenya and Ivory Coast in Africa respectively. United States was having the highest number of sampling locations in North America. Comparatively, the occurrences of Montagnula species using metabarcoding data in China, the USA, and European countries are relatively well-documented. However, the rest of the world remains a mystery in terms of Montagnula distribution. For example, the majority of Asia, including India and Russia, lacks metabarcoding data for Montagnula species. This emphasizes the need for more extensive research and data collection to better understand the global distribution of Montagnula and its ecological roles.

The authors have declared that no competing interests exist.

No ethical statement was reported.

Dhanushka Wanasinghe thanks CAS President’s International Fellowship Initiative (number 2021FYB0005), the National Science Foundation of China (NSFC) under the project code 32150410362, Smart Yunnan Project (Young Scientists) under project code E13K281261 and the Postdoctoral Fund from Human Resources and Social Security Bureau of Yunnan Province. Thilina Nimalrathna expresses gratitude for the support provided by the Belt and Road Chinese Government Scholarship and The Alliance of International Science Organizations (ANSO) Ph.D. scholarship. We also extend our appreciation to the Researchers Supporting Project at King Saud University, Riyadh, Saudi Arabia, for funding this research project (Fund no. RSP2024R487). Jianchu Xu thanks National Natural Science Foundation of China (grant number: 31861143002), the Yunnan Provincial Science and Technology Department (grant number: 202101AS070045), Yunnan Provincial Science and Technology Department (grant number: 202205AM070007) and Yunnan Department of Sciences and Technology of China (grant number: 202302AE090023).

Conceptualization: DNW. Data curation: LQX, DNW. Formal analysis: TKF, DNW, TSN. Investigation: TSN, DNW. Methodology: TSN, DNW. Project administration: PEM, JX. Resources: JX. Supervision: JX, PEM. Writing – original draft: TSN, DNW. Writing – review and editing: PEM, TKF.

Dhanushka N. Wanasinghe https://orcid.org/0000-0003-1759-3933

Thilina S. Nimalrathna https://orcid.org/0000-0002-2368-042X

Li Qin Xian https://orcid.org/0009-0006-4936-9409

Turki KH. Faraj https://orcid.org/0000-0002-6012-8474

Jianchu Xu https://orcid.org/0000-0002-2485-2254

Peter E. Mortimer https://orcid.org/0000-0003-3188-9327

All of the data that support the findings of this study are available in the main text or Supplementary Information.