Taxonomy and phylogenetic appraisal of Spegazzinia musae sp. nov. and S. deightonii (Didymosphaeriaceae, Pleosporales) on Musaceae from Thailand

Abstract Tropical plants host a range of fungal niches including endophytes, pathogens, epiphytes and saprobes. A study undertaken to discover the saprobic fungal species associated with Musa sp. (banana) from northern Thailand found two hyphomycetous taxa of Spegazzinia (Didymosphaeriaceae, Pleosporales). These were collected during the dry season and their morpho-molecular taxonomic relationships were investigated. Based on phylogenetic analysis of combined SSU, LSU, ITS and TEF1-α sequence data (77% ML, 0.99 BYPP) and contrasting morphological features to the sister taxon, we introduce Spegazzinia musae as a novel species from a decaying leaf of Musa sp. Details on the taxonomy, ecology and geographical distribution of Spegazzinia species are provided. In addition, we report S. deightonii as a new host record from Musa sp. Our data further validate the taxonomic placement of Spegazzinia in Didymosphaeriaceae.

Spegazzinia was established by Saccardo (1880) based on S. ornata. Currently 17 taxa are listed in Species Fungorum (2020). Based on morphology, the genus was placed in Apiosporaceae (Sordariomycetes) by Hyde et al. (1998). Based on SSU, LSU, ITS and TEF1-α sequence data of S. deightonii and S. tessarthra, Tanaka et al. (2015) placed Spegazzinia in Didymosphaeriaceae (Dothideomycetes). This was supported by a phylogenetic analysis which placed Spegazzinia in a basal clade in Didymosphaeriaceae . Hughes (1953) characterized Spegazzinia as a hypomycetous taxon with a unique basauxic conidiophore ontogeny (conidiophores that arise and elongate from a cupshaped basal cell called a conidiophore mother cell). The conidia of Spegazzinia are brown to dark brown and dimorphic in most species, with a disc-shaped form and a stellate form (Ellis 1971;Manoharachary and Kunwar 2010). However, little molecular data for this genus is available in the GenBank (https://www.ncbi.nlm.nih.gov/). Therefore, for a better phylogenetic resolution of the genus in Didymosphaeriaceae, the previously identified taxa should be recollected to obtain DNA sequence data and morphological descriptions.
In this present study, we introduce Spegazzinia musae sp. nov. and report the first occurrence of Spegazzinia deightonii from Musa sp. in Thailand. We provide detailed morphological descriptions, illustrations and molecular justification for the introduction of Spegazzinia musae sp. nov. Our molecular analyses further support the phylogenetic placement of Spegazzinia in Didymosphaeriaceae.

Sample collection, morphological studies and isolation
Dead plant materials of Musa sp. (banana) were collected from Thailand during the dry season of 2018 to 2019. Specimens were transferred to the laboratory in cardboard boxes. Samples were examined with a Motic SMZ 168 Series microscope. Powdery masses of conidia were mounted in water for microscopic studies and photomicrography. The taxa were examined using a Nikon ECLIPSE 80i compound microscope and photographed with a Canon 550D digital camera fitted to the microscope. Measurements were made with the Tarosoft (R) Image Frame Work program and images used for figures processed with Adobe Photoshop CS3 Extended version 10.0 software (Adobe Systems, USA).
Single spore isolation was carried out following the method described in Chomnunti et al. (2014). Germinated spores were individually transferred to potato dextrose agar (PDA) plates and grown at 25 °C in daylight. Colony characteristics were observed and measured after 3 weeks. Specimens were deposited in the Mae Fah Luang University (MFLU) Herbarium, Chiang Rai, Thailand. Living cultures were deposited in the Culture Collection of Mae Fah Luang University (MFLUCC).

DNA extraction and PCR amplification
Fungal isolates were grown on PDA for 4 weeks at 25 °C and total genomic DNA was extracted from 50 to 100 mg of axenic mycelium of the growing cultures according to Wanasinghe et al. (2018). The mycelium was ground to a fine powder with liquid nitrogen and fungal DNA was extracted using the Biospin Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux, P.R. China) according to the instructions of the manufacturer. Four gene regions, the internal transcribed spacer (ITS), partial 18S small subunit (SSU), partial 28S large subunit (LSU), and partial translation elongation factor 1-alpha gene (TEF1-α) were amplified using ITS5/ITS4 (White et al. 1990), NS1/NS4 (White et al. 1990), LR0R/LR5 (Vilgalys and Hester 1990) and EF1-983F/EF1-2218R (Rehner 2001) primers, respectively.
Polymerase chain reaction (PCR) was conducted according to the following protocol. The total volume of the PCR reaction was 25 μL containing 12.5 μL of 2 × Power Taq PCR MasterMix (a premix and ready to use solution, including 0.1 Units/ μL Taq DNA Polymerase, 500 μm dNTP Mixture each (dATP, dCTP, dGTP, dTTP), 20 mM Tris-HCL pH 8.3, 100 mM KCl, 3 mM MgCl 2 , stabilizer and enhancer), 1 μL of each primer (10 pM), 2 μL genomic DNA template and 8.5 μL double distilled water (ddH 2 O). The reaction was conducted by running for 40 cycles. The annealing temperature was 56 °C for ITS and LSU, 57.2 °C for TEF1-α and 55 °C for SSU and initially 95 °C for 3 mins, denaturation at 95 °C for 30 seconds, annealing for 1 min, elongation at 72 °C for 30 seconds, and final extension at 72 °C for 10 mins for all gene regions. PCR amplification was confirmed on 1% agarose electrophoresis gels stained with ethidium bromide. The amplified PCR fragments were sent to a commercial sequencing provider (TsingKe Biological Technology (Beijing) Co., Ltd, China). The nucleotide sequence data acquired were deposited in GenBank.

Sequencing and sequence alignment
Obtained sequences were subjected to BLASTn search in GenBank (https://blast.ncbi. nlm.nih.gov/Blast.cgi). BLASTn search results and initial morphological studies supported that our isolates belonged to Didymosphaeriaceae. Other sequences used in the analyses were obtained from GenBank based on recently published data Jayasiri et al. 2019) (Table 1). The single gene alignments were automatically done by MAFFT v. 7.036 (http://mafft.cbrc.jp/alignment/server/index.html, Katoh et al. 2019) using the default settings and later refined where necessary, using BioEdit v. 7.0.5.2 (Hall 1999). The finalized alignment and tree were submitted to TreeBASE (submission ID: 25686, http://www.treebase.org/).

Phylogenetic analysis
Maximum likelihood (ML) trees were generated using the RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis et al. 2008;Stamatakis 2014) in the CIPRES Science Gateway platform (Miller et al. 2010) using GTR+I+G model of evolution. Bootstrap support was obtained by running 1000 pseudo-replicates. Maximum likelihood bootstrap values (ML) equal or greater than 60% are given above each node in blue (Figure 1).
A Bayesian inference analysis was conducted with MrBayes v. 3.1.2 (Huelsenbeck and Ronquist 2001) to evaluate posterior probabilities (PP) (Rannala and Yang 1996;Zhaxybayeva and Gogarten 2002) by Markov chain Monte Carlo sampling (BMCMC). Two parallel runs were conducted, using the default settings, but with the following adjustments: four simultaneous Markov chains were run for 2,000,000 generations and trees were sampled every 100 th generation and 20,000 trees were obtained. The first 4,000 trees, representing the burning phase of the analyses were discarded. The remaining 16,000 trees were used for calculating PP in the majority rule consensus tree. Branches with Bayesian posterior probabilities (BYPP) greater

Data resources
The data underpinning the analysis reported in this paper are deposited in the Dryad Data Repository at https://doi.org/10.5061/dryad.2ngf1vhk6.
Culture characteristics. Conidia germinating on PDA within 12-15 h, germ tubes produced from one or several cells. Colonies growing on PDA, reaching a diameter of 46 mm after 14 d at 25 °C, greyish white, unevenly raised, surface rough, moderately dense, radially striated at center, margin crenulate; reverse white to greyish white.
Notes. Based on BLASTn search results of SSU, LSU, ITS and TEF1-α sequence data, Spegazzinia musae showed a high similarity (SSU = 98.24%, LSU = 98.92%, ITS = 96.91%, TEF1-α = 98.11%) to S. neosundara . In the multigene phylogeny, S. musae groups as a sister taxon to S. deightonii with strong statistical support (77% ML, 0.99 BYPP) (Figure 1). Also, ITS sequence comparison revealed 3.75% base pair differences between S. musae and S. deightonii, which is in agreement with the species concept outlined by Jeewon and Hyde (2016). Besides, S. musae has contrasting morphological features to S. deightonii in both kinds of conidia. The diskshaped conidia of S. musae are 4-celled and do not bear spines at the periphery of cells, while the disc-shaped conidia of S. deightonii are 8-celled and spiny. Based on contrasting morphological differences and significant statistical support from our molecular phylogeny, Spegazzinia musae is introduced as a new species.

Discussion
Spegazzinia is ubiquitous in the environment. Several taxa of Spegazzinia occur as saprobes on dead material of tropical, subtropical and temperate vascular plants (Ellis 1971;Subramanian 1988;Caretta et al. 1999;Delgado-Rodríguez et al. 2002;Bhat 2010;Leão-Ferreira and Gusmão 2010;Manoharachary and Kunwar 2010). In addition, Spegazzinia was also recorded from soil (Ellis 1971), dredged sediments of marine and brackish estuaries (Borut and Johnson 1962) and grassland vegetation (Caretta et al. 1999). Spegazzinia tessarthra was recorded as an endophyte from lichens (Manish et al. 2014) and recently S. bromeliacearum was introduced as an endophyte from the leaves of Tilandsia catimbauensis . Damon (1953) considered S. tessarthra to be an important decomposer of monocotyledonous plants and other cellulose containing materials in tropical and subtropical areas. Spegazzinia deightonii was previously recorded on monocotyledons such as Areca catechu (China, Taiwan;Matsushima 1980), Cocos nucifera (China; Tianyu et al. 2009) and Panicum maximum (Hong Kong; Lu et al. 2000) (Farr and Rossman 2020). Our study presents the first report of Spegazzinia deightonii in Musaceae as a saprobe and introduces our new species, S. musae.
There does not appear to be any host-specificity as the genus is found on a wide range of hosts in various habitats and there are no records of a pathogenic lifestyle. Some Spegazzinia species (such as S. tessarthra) have been identified as saprobes and endophytes and therefore the genus may have the potential of switching nutritional modes during the degradation of plant material (Promputtha et al. 2007).
Spegazzinia is a unique taxon among other dematiaceous hyphomycetes due to its conidial morphology and basauxic conidiogenesis. Most Spegazzinia species have contrasting morphological features in the shapes of α and β conidia. Some taxa bear spines in both types of conidia while some taxa do not bear spines. Simultaneously, some species of Spegazzinia such as S. radermacherae, S. tessarthra show similar characters in morphology apart from dimensions of conidia. The length of conidiophores can be varied with the environmental stresses (Cole 1974). Therefore, the use of morphological data coupled with DNA sequence data (SSU, LSU, ITS and TEF-α) will be crucial for better taxonomic resolutions in this genus.
Microfungal studies in Musa sp. are mostly oriented towards pathogens and endophytes due to the economic value of the fruit crop. Most of the pathogenic species descriptively studied from Musa sp. are identified as Colletotrichum, Fusarium, Mycosphaerella, Neocordana and Phyllosticta (Giatgong 1980;Wulandari et al. 2010;Churchill 2011;Guarnaccia et al. 2017;Marin-Felix et al. 2019;Maryani et al. 2019). The endophytic fungal populations of Musa sp. were studied by Brown et al. (1998), Photita et al. (2001aPhotita et al. ( , 2004 and Samarakoon et al. (2019). Few studies have documented the saprobic diversity of Musa sp. and as we believe that there are saprobic niches associated with Musa sp. that are still unrevealed, taxonomists should investigate this hidden diversity for conservation purposes.