Morphology and Phylogeny Reveal Three Montagnula Species from China and Thailand

Four stains were isolated from two fresh twigs of Helwingia himalaica and two dead woods during investigations of micro-fungi in China and Thailand. Phylogenetic analyses of four gene regions LSU, ITS, SSU and tef1-α revealed the placement of these species in Montagnula. Based on the morphological examination and molecular data, two new species, M. aquatica and M. guiyangensis, and a known species M. donacina are described. Descriptions and illustrations of the new collections and a key to the Montagnula species are provided. Montagnula chromolaenicola, M. puerensis, M. saikhuensis, and M. thailandica are discussed and synonymized under M. donacina.


Introduction
Didymosphaeriaceae (Pleosporales) was established by Munk [1] and with Didymosphaeria as the type genus. There are 33 genera accepted in this family based on morphology and phylogenetic analyses [2,3]. Species belonging to Didymosphaeriaceae have a wide geographical and host distribution and have different modes of nutrition, such as saprobic on plant litter, herbaceous stems, or in soil; endophytic on healthy leaves or twigs; and pathogenic on plants, animals, or humans [2,[4][5][6][7][8][9].
To study the taxonomy and diversity of Montagnula species, four Montagnula specimens were obtained from terrestrial and freshwater habitats in China and Thailand. Based on the morphological examination and phylogenetic analyses, two new species, viz. M. aquatica and M. guiyangensis, and a known species, M. donacina are introduced with illustrations and descriptions. We also provide a key to Montagnula species.
The ML and BYPP trees (not shown) were similar in topology. The genus Montagnula formed an independent topmost clade in the phylogenetic tree. Montagnula species were divided into four clades in the phylogenetic tree. Our four strains nested within the genus and represented three species. Culture characteristics: Ascospores germinated on PDA within 12 h at 25 • C. Germ tubes produced from both ends. Colonies on PDA reached 5 cm diam. after 3 weeks at 25 • C; mycelium white, flossy, circular, with the entire edge; white to yellow in reverse.

Figure 1.
The ML tree based on a combined dataset of LSU, ITS, SSU, and tef1-α sequence data. The tree was rooted with Fuscostagonospora sasae (HHUF 29106) and F. cytisi (MFLUCC 16-0622). Bootstrap support values for ML greater than 75% and Bayesian posterior probabilities greater than 0.95 are given near the nodes, respectively. Ex-type strains are in bold, the new isolates are in red.
Culture characteristics: Ascospores germinated on PDA within 12 h at 25 • C. Germ tubes produced from both ends. Colonies on PDA reached 7 cm diam after four weeks at 25 • C, mycelium white to gray, flossy, circular, undulate, yellow in reverse.

Discussion
Montagnula species have a worldwide distribution that has been reported from America, Australia, Bahamas, China, Italy, Portugal, and Thailand [8,21]. Previous literature reported that all Montagnula species have been derived from terrestrial habitats [7,8,10,11,20,21,23]. We introduced a freshwater Montagnula species here that broke the record of the monolithic habitat for Montagnula species. These species have various hosts, such as Agave sp., Pandanus sp., and Ilex sp. [5,21]. However, rarely have studies focused on fungi associated with H. himalaica (Helwingiaceae). Helwingia himalaica is distributed in Bhutan, China, Nepal, and Thailand (https://www.havlis.cz/karta_en. php?kytkaid=5087 accessed on 27 January 2023). It has a high medicinal value that is used to treat colds, coughs, stomach pains, and fractures. In this study, we introduced a new species, M. guiyangensis, which was isolated from H. himalaica.

Collection, Examination, and Isolation
The fresh samples were collected in China and Thailand from 2019 to 2022. Samples were brought to the laboratory in Ziplock plastic bags for examination, as described in Senanayake et al. [29]. The fruiting bodies on natural substrates were observed and photographed using a stereomicroscope (SteREO Discovery, V12, Carl Zeiss Microscopy GmBH, Berlin, Germany; VHX-7000, Keyence, Osaka, Japan). Morphological characters were observed using a Nikon ECLIPSE Ni compound microscope (Nikon, Tokyo, Japan) photographed with a Nikon DS-Ri2 digital camera (Nikon, Japan), and Carl Zeiss compound microscope (Carl Zeiss AG, Germany) photographed with an Axiocam 208 color digital camera (Carl Zeiss AG, Germany). The photo plates were made by the Adobe Photoshop CS6 Extended v. 13.0 software. Measurements were done with the Tarosoft (R) Image Frame Work Version 0.9.7 software.
Single spore isolation was used to obtain pure cultures following the methods described by Senanayake et al. [29]. Germinated ascospores were transferred to new potato dextrose agar (PDA) plates and incubated at 25 • C for 4 weeks. The pure cultures obtained were deposited in Mae Fah Luang University Culture Collection (MFLUCC), Chiang Rai, Thailand, and the Guizhou University Culture Collection (GUCC), Guiyang, China. Herbaria materials were deposited in the herbarium of Mae Fah Luang University (MFLU), Chiang Rai, Thailand, and the Kunming Institute of Botany Academia Sinica (HKAS), Kunming, China. Facesoffungi (FoF) and Index Fungorum numbers were acquired as described in Jayasiri et al. [30] and Index Fungorum (2023) [31]. Records were added to the Mekong Subregion (GMS) database [32]. The establishment of new species was decided upon the recommendations of Chethana et al. [27] and Jayawardena et al. [33].

DNA Extraction, PCR Amplification, and Sequencing
PrepManTM Ultra Sample Preparation Reagent (Thermo Fisher Scientific, Yokohama, Japan) was used to extract DNA directly from fruiting bodies. BIOMIGA Fungus Genomic DNA Extraction Kit (Biomiga, San Diego, CA, USA) was used to extract DNA from fresh fungal mycelia, which were grown on PDA medium for 4 weeks at 25 • C. Three genes were selected in this study: the large subunit nuclear ribosomal DNA (LSU), the small subunit nuclear ribosomal DNA (SSU), the internal transcribed spacers (ITS), and the translation elongation factor 1 (tef1-α). Polymerase chain reaction (PCR) was carried out in 20 µL reaction volume, which contained 10 µL 2 × PCR Master Mix, 7 µL ddH 2 O, 1 µL of each primer, and 1 µL template DNA. The PCR thermal cycle program and primers are given in Table 5. Purification and sequencing of PCR products were carried out at SinoGenoMax (Beijing) Co., China.
The BYPP analyses were conducted in CIPRES [40] with MrBayes on XSEDE 3.2.7a [42]. The best nucleotide substitution model for each data partition was evaluated by MrModeltest 2.2 [43]. The substitution model GTR+I+G was decided for LSU, ITS, and SSU sequences. The Markov chain Monte Carlo (MCMC) sampling approach was used to calculate posterior probabilities (PP) [44]. Six simultaneous Markov chains were run for 10 million generations and trees were sampled every 1000th generation. The first 20% of trees, representing the burn-in phase of the analyses, were discarded and the remaining trees were used for calculating the PP value in the majority rule consensus tree.
Phylogenetic trees were viewed using FigTree v1.4.0 [45] and modified in Microsoft Office PowerPoint 2019 and converted to a jpg file using Adobe Photoshop CS6 Extended 10.0 (Adobe Systems, San Jose, CA, USA). The new sequences derived from this study were deposited in GenBank. 17