Taxonomy and phylogeny of saprobic Ascomycota on Poaceae and worldwide checklist of Ascomycota on grasses

Abstract

Grasses include three plant families viz. Cyperaceae, Juncaceae, and Poaceae. Amongst the three families, Poaceae are well-known grasses. The history of the studies on grass fungi dates back to the early 1900s. Hence, grass fungal studies have a long history. Most of the grass fungal records are scattered in the literature. Several lists are available for several grass groups, commercial grasses, or selected fungal taxa, which shows the necessity of providing a concise, systematic literature collection on grass fungi. Further, there is no proper document to understand the current status of grass fungi, such as; how many species have been reported, how many valid taxa are there, and how much diversity is there. Hence, a checklist on Ascomycetous grass fungi is provided to give up-to-date knowledge on grass fungal diversity. The data were retrieved from well-accepted lists such as Saccardo sylloge, Petrak’s index, Index Fungorum, and taxonomically and phylogenetically accepted research publications. Further, Fungal Databases like U.S. National Fungus Collections ARS/ USDA (https://nt.ars-grin.gov/fungaldatabases/) were also used to obtain the data. The retrieved fungal names were checked on the Index Fungorum and MycoBank for the validity of the taxa. The currently accepted name in the Species Fungorum was given as the latest name. The checklist provides a good insight into the current knowledge on Ascomycetous fungal diversity on grasses (Cyperaceae, Juncaceae, Poaceae). The highest number of species are reported from Dothideomycetes (1340 species), while the Sordariomycetes, Leotiomycetes, and Ascomycota incertae sedis have 990, 327, and 267 species, respectively. In addition, species from Arthoniomycetes, Eurotiomycetes, Lecanoromycetes, Orbiliomycetes, Pezizomycetes, and Saccharomycetes are also reported. In Dothideomycetes, the highest number of species are reported from Pleosporales (845 species). The majority of Sordariomycetes are represented by Hypocreales (262 species) and Xylariales (144 species). The highest number of Ascomycetous fungi are represented by Pleosporales (Dothideomycetes), Hypocreales (Sordariomycetes), and Xylariales (Sordariomycetes). Phyllachora, with 78 species, has the highest diversity. High generic diversity was observed in Mycosphaerellaceae (30 genera) and Phaeosphaeriaceae (20 genera).

For the taxonomic and phylogenetic study of saprobic Ascomycetes on Poaceae, Samples were collected from three collection sites viz. Nothern Thailand, Thailand; Kunming, Yunnan province and Chiyayi, Taiwan region, China. A total of 300 samples were collected randomly from all sites (100 samples from each site), and 200 cultures were obtained. Among them, 150 isolates were identified and reported. In Thailand, the majority of the samples were collected from Saccharum sp., Pennisetum sp., and Arundo sp. In Kunming, Yunnan province, the majority of samples were collected from Phragmites sp., while, in the Taiwan region, the majority were from Miscanthus sp., Pennisetum sp., and Setaria sp. The macro and micro-morphology of the fungi were observed and recorded. Fungi were isolated using the single spore isolation technique. Fungal DNA was extracted using fresh mycelium or the fruiting structures. PCR amplification was performed for the SSU, LSU, ITS, TEF, and RPB2 gene regions. Concatenated sequences were used to construct the phylogenetic trees. ML, MP, and BYYP analyses were used for the phylogenetic relatedness studies. In the current study, the genera/ Alishanica Karun., C.H. Kuo & K.D. Hyde, Kwanghwana Karun., C.H. Kuo & K.D.Hyde and Yunnanensis A. Karunarathna, Phookamsak & K. D. Hyde are introduced. In addition, novel species, Alishanica miscanthi Karun., C.H. Kuo & K.D. Hyde, Apiculospora penniseti A. Karunarathna & C.H. Kuo, Chloridium macrocladum ((Sacc.) S. Hughes) Karun., Maharachch., C.H.Kuo & K.D.Hyde, Kwanghwana miscanthi A. Karunarathna, C. H. Kuo & K. D. Hyde, Neophaeosphaeria phragmiticola A. Karunarathna & K.D. Hyde, Neoroussoella alishanensis A. Karunarathna, C.H. Kuo, Phookamsak & K.D. Hyde, Neosetophoma miscanthi Karun., C.H. Kuo & K.D. Hyde, Phaeopoacea muriformis A. Karunarathna & K.D. Hyde, Sparticola muriformis A. Karunarathna & Phookamsak, and Yunnanensis phragmitis A. Karunarathna, Phookamsak & K. D Hyde are described and introduced.

Further, new hot and geographical records are provided for the, Bipolaris yamadae (Y. Nisik.) Shoemaker, Epicoccum nigrum Link, Mag., Neokalmusia didymospora D.Q. Dai & K.D. Hyde, Occultibambusa bambusae D.Q. Dai & K.D. Hyde, Phaeosphaeria chinensis K.K. Zhang, Hongsanan, Tennakoon & N. Xie, Roussoella siamensis Phook., Jian K. Liu & K.D. Hyde, Stictis urceolata (Ach.) Gilenstam (Ach.) Gilenstam, Tetraploa sasicola (Kaz. Tanaka & K. Hiray.) Kaz. Tanaka & K. Hiray. and Tetraploa sasicola (Kaz. Tanaka & K. Hiray.) Kaz. Tanaka & K. Hiray

Based on the current study, the highest fungal diversity was reported from Dothidimycetes (73 records). In Dothidiomycetes, Phaeosphaeriaceae is the highest recorded family (28 records). All collection areas showed a high number of Phaeosphaeriaceae records. Further, Tetraplosphaeriaceae, Pleosporaceae, and Nectriaceae have high number of records. In this study, species from Apiosporaceae, Apiosporaceae, Arthopyreniaceae, Ascomycota incertae sedis, Botryosphaeriaceae, Cainiaceae, Chaetosphaeriaceae, Coniothyriaceae, Diaporthaceae, Dictyosporiaceae, Didymellaceae, Glomerellaceae, Helotiales Incertae sedis, Hypoxylaceae, Mycosphaerellaceae, Niessliaceae, Occultibambusaceae, Pestalotiopsidaceae, Pleosporales, Pyriculariaceae, Roussoellaceae, Shiraiaceae, Stictidaceae, Thyridariaceae, Torulaceae, and Xylariaceae are also reported. However, higher fungal diversity has been shown in the Taiwan region of China compared to the other collection sites. A comparatively higher number of Sordariomycetes records are reported in the Taiwan region, compared to the other collection areas. Even though, the current study is on saprobic fungi, the results agree with the checklist. High fungal diversity was observed on Dothidiomycetes. Further, the majority of fungal records were from Phaeosphaeriaceae.

Fungi show two different reproductive modes: asexual reproduction and sexual reproduction. Both states are available in their natural habitats. For the proper taxonomic nomenclature, understanding each reproductive state of the same fungi is important to satisfy the "one fungus one name rule” which avoids the erroneous nomenclature of the same fungi under two different names. For such purposes, a modern phylogenetic approach together with the pair-wise comparison of base pairs in selected gene regions can be used. In the current study, sexual and asexual morphs of fungi were collected separately from their natural habitats. The sequence data for each fungus was obtained from PCR followed by Sanger sequencing. All the sequences were subjected to ML, MP, and BYYP, based phylogeny. The closely clustered different reproductive modes were selected for further analyses. The base pairs of each gene region were compared using the “global alignment” facility in NCBI GenBank. The strains with the similarity above 98% were selected and the morphology of each state was compared with the other species in the genus and the family. With the help of morphology, phylogeny, and base-pair comparison, the sexual asexual link was established. In this study, the genus Yunnanensis on Poaceae from Yunnan Province, in China, is introduced with sexual and asexual morphs isolated from its natural habitat. The Yunnanensis is introduced to accommodate Y. phragmitis. Yunnanensis is morphologically distinct from other genera in Phaeosphaeriaceae in having ascomata with evenly thickened walls comprising textura angularis and textura angularis to textura prismatica inner layers, broadly cylindrical to cylindric-clavate asci, muriform ascospores with a mucilaginous sheath. However, a coelomycetous asexual state with muriform conidia. The phylogeny also supports Yunnanensis as a novel genus basal to Neosetophoma. However, in the phylogenetic study, sexual and asexual morphs are claded together with high statistical supports. Further, the base pair comparison confirmed that the two different morphs belong to the same species. In another collection from the Taiwan region of China, a new species of Neoroussoella (Roussoellaceae) is introduced with asexual and sexual morphs. Both morphs were isolated from their natural habitats and Neoroussoella alishanense is introduced based on Taxonomy, phylogeny, and the base pair comparison.

The current study provides proper systematic documentation of Ascomycetes associated with grasses. Further, the study provides novel fungal species, novel geographic and host records, novel herbarium collections, fungal culture collections and molecular data to broaden the current knowledge of grass fungal diversity.