Monilochaetes pteridophytophila (Australiascaceae, Glomerellales), a new fungus from tree fern

Abstract Background During taxonomic and phylogenetic studies of fungi on pteridophytes in Thailand, Monilochaetes pteridophytophila sp. nov. was collected from the frond stalks of a tree fern (Alsophila costularis, Cyatheaceae). The new species is introduced, based on evidence from morphology and phylogenetic analyses of a concatenated dataset of LSU, ITS, SSU and RPB2 sequences. New information Monilochaetes pteridophytophila differs from extant species of Monilochaetes in having darker conidiophores with fewer septae (1–4-septate). Monilochaetes pteridophytophila forms a distinct clade, basal from other species of Monilochaetes in Australiascaceae. A detailed description and illustrations of the new species are provided. We also provided a synopsis of accepted species of Monilochaetes.


Introduction
Studies on the diversity of fungi on pteridophytes have revealed many new taxa during the last decade (Mehltreter 2010, Braun et al. 2013, Kirschner and Liu 2014, Guatimosim et al. 2016, Kirschner et al. 2019). An estimated 670 species of fern occur in Thailand (Lindsay and Middleton 2009), making it a suitable area for studying the fungi associated with ferns. However, the study of fungi on ferns is in its infancy (Razikin et al. 2014, Kirschner et al. 2019. Cyatheaceae, a family of scaly tree ferns in Cyatheales, is widely distributed in tropical and subtropical areas (Lehnert 2011, Korall andPryer 2014). Species of Cyatheaceae diverged ca. 150 (146-168) million years ago during the Late Jurassic period (Korall and Pryer 2014). Many taxa in this family are threatened species, including Cyathea brunoniana, C. gigantea and C. henryi ( Balkrishna et al. 2020, Coritico andAmoroso 2020).
Monilochaetes Halst. ex Harter was introduced by Harter (1916) to accommodate a pathogenic fungus, M. infuscans Harter, that caused scurf disease of the sweet potato. Monilochaetes infuscans was first reported by Halsted (1890), but the species is considered invalid due to the lack of morphological description and illustrations. Réblová et al. (2011a) established the family Australiascaceae Réblová & W. Gams to accommodate Australiasca Sivan. & Alcorn (as a sexual morph) and Monilochaetes (as an asexual morph). Sivanesan and Alcorn (2002) introduced Australiasca with A. queenslandica Sivan. & Alcorn as the type species, which was linked to Dischloridium camelliae Alcorn & Sivan as an asexual morph. Réblová et al. (2011a) treated Dischloridium B. Sutton as the generic synonym of Monilochaetes, based on phylogenetic analysis of ITS and LSU sequences. Following the "One Fungus One Name" (1F1N) principle, Australiasca was synonymised under Monilochaetes, the latter being older (Réblová et al. 2016, Hyde et al. 2020a (Sivanesan and Alcorn 2002, Réblová et al. 2011a, Réblová et al. 2011b, Zhou et al. 2017, Crous et al. 2018.
In this study, a new species of Monilochaetes, M. pteridophytophila, is described, illustrated and compared with closely-related taxa. Morphological study and multilocus phylogenetic analyses confirm the identity of the new species and confirm its placement in Monilochaetes.

Sample collection, isolation and conservation
Frond stalks of Alsophila costularis (tree fern) were collected in a disturbed forest near the roadside in Tak Province, Thailand. Specimens were packed into a plastic bag for transportation to the laboratory and the associated metadata were noted (date, locality and host). Fungal colonies on the host surface were observed and examined using a stereomicroscope (Leica EZ4, Leica Microsystems AG, Singapore). Micro-morphological characters were documented with a Nikon DS-Ri2 digital camera fitted to a Nikon ECLIPSE Ni compound microscope (Nikon, Japan). Measurements of morphological structures (conidiophores, conidiogenous cells and conidia) were made with the Tarosoft (R) Image Frame Work. Figures were processed and combined with Adobe Illustrator CS6 (Adobe Systems, USA).
Single spore isolation was carried out to obtain a pure culture, following the method described by . Germinated conidia were aseptically transferred to potato dextrose agar (PDA) plates and incubated at 25°C. Cultures were grown for 2 weeks and culture characteristics, such as size, shape, colour and texture, were recorded. The holotype specimen and ex-type living culture are deposited in the Herbarium of Mae Fah Luang University (MFLU) and Mae Fah Luang University Culture Collection (MFLUCC), Chiang Rai, Thailand, respectively. An isotype specimen is deposited at the Herbarium of Guizhou Academy of Agricultural Sciences (GZAAS), Guiyang, China.

DNA extraction, PCR amplification and sequencing
Fresh fungal mycelium grown on PDA at 25°C for 2 weeks was used to extract DNA. Genomic DNA was extracted by using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux, China), following the manufacturer's instructions. We amplified the internal transcribed spacer (ITS) region, the small and large subunits of the ribosomal RNA gene (SSU, LSU) and the second largest subunit of RNA polymerase II (RPB2). Primer pairs and PCR thermal cycle conditions are listed in Table 1. The quality of PCR products was checked on 1% agarose gel electrophoresis stained with ethidium bromide. Successful PCR products were sent to Sangon Biotech (Shanghai, China) for purification and sequencing. Forward and reverse sequence reads were assembled using SeqMan v. 7.0.0 (DNASTAR, Madison, WI). Consensus sequences were submitted to NCBI GenBank (Table 2).
Taxa used to infer the phylogenetic tree and their GenBank accession numbers.
Notes: "-" as meaning no data available in GenBank. The newly-generated sequences are underlined. The ex-type strains are in bold.
Culture characteristics: Conidia germinating on PDA within 12 hours at 25℃, with hyaline germ tube germinating from the base of conidia. Colonies growing on PDA at 25℃, circular, flat surface, planar, thin, dark brown, reaching 2 cm diam. in 7 days, edge entire, emission at margin, dark brown to pale brown in reverse from the centre to margin of the colony.

Etymology
Referring to the host, which is a pteridophyte.

Analysis
Analysis Ⅰ: Phylogenetic reconstruction of a combined LSU, ITS, SSU and RPB2 sequence dataset The aligned, concatenated sequence matrix comprised sequence data for 39 taxa from seven families of the following loci: LSU (853 bp), ITS (489 bp), SSU (1,014 bp) and RPB2 (1,061 bp). Included sequences represented taxa of Glomerellales and three outgroup taxa, Collariella bostrychodes (CBS 586.83), Corynascus fumimontanus (CBS 137294) and Leptosillia pistaciae (CBS 128196). The sequence matrix comprised 3,417 characters (including gaps), of which 2,317 characters were constant, 185 variable characters were parsimony-uninformative and 915 characters were parsimony-informative. The matrix had 1,188 distinct alignment patterns, with 40.80% undetermined characters or gaps. The ML and BI analyses of the concatenated LSU-ITS-SSU-RPB2 dataset resulted in similar tree topologies (Fig. 2).
The phylogenetic tree shows that all strains of Monilochaetes clustered within Australiascaceae. The new species M. pteridophytophila forms a distinct clade, basal to other species of Monilochaetes with BS = 98% MLBS and PP = 1.00 (Fig. 2).

Monilochaetes basicurvata
Palm petiole    Bhat and Kendrick (1993) Monilochaetes pteridophytophila is the second species found on a tree fern; M. laeensis occurs on tree ferns in Australia and the UK (Kirk 1986, Réblová et al. 2011a. The tree topologies resulting from the phylogenetic reconstruction of a combined LSU-ITS dataset (analysis Ⅱ, Suppl. material 1) and the concatenated LSU-ITS-SSU-RPB2 dataset (analysis Ⅰ, Fig. 2) were overall similar and not significantly different. A comparison of phylogenetic analysis Ⅰ and Ⅱ with the analysis by Hyde et al. (2020a) showed negligible variation in tree topologies in Glomerellales, even with the inclusion of SSU and RPB2 data. The phylogeny in the current study suggests that LSU and ITS sequences can resolve interspecific relationships within Monilochaetes, as well as interfamilial relationships within Glomerellales.