Neomonodictysaquatica sp. nov. (Pleurotheciaceae) from a plateau lake in Yunnan Province, China

Abstract Background In this study, a new species Neomonodictysaquatica was collected from submerged decaying wood in Erhai Lake, Yunnan Province, China. New information Neomonodictysaquatica is characterised by acrogenous, solitary, oval, dictyospores (most are transverse septum, occasionally vertical septum, in immaturity the septum is clear, but when mature, the conidia becomes darker so the septum is not clear), smooth-walled conidia. The immature conidia are usually hyaline to olivaceous and mature conidia are usually darkened to black, sometimes with one pale basal cell. Phylogenetic analyses of combined ITS and LSU sequence data showed that the new collection is distinct from other Neomonodictys species. Description and illustration are provided as well.


Introduction
Pleurotheciales was introduced by Réblová et al. (2016), based on morphological characters and phylogenetic analyses. Members of Pleurotheciales are mostly saprobic on wood ) and some species have been identified as opportunistic human pathogens (Guarro et al. 2000, Chew et al. 2010, Réblová et al. 2020. Species of the order were collected on submerged decaying wood in lentic and lotic habitats in temperate, subtropical and tropical zones in Asia, Europe, Melanesia and North America (Matsushima 1971, Réblová et al. 2012, Réblová et al. 2016, Réblová et al. 2020, Hernández-Restrepo et al. 2017, Luo et al. 2018a).
Pleurotheciaceae is a single family of Pleurotheciales. It is typified by Pleurothecium with P. recurvatum as the type species (Morgan) Höhn (Réblová et al. 2016). Recently,  updated the phylogenetic tree for Pleurotheciales and introduced a new genus Neomonodictys Y.Z. Lu, C.G. Lin & K.D. Hyde. Currently, ten genera are accepted in this family (Réblová et al. 2016, Maharachchikumbura et al. 2016, Hernández-Restrepo et al. 2017, Goh and Kuo 2021. The monophyletic asexual genus Neomonodictys is established for a fungus (Neomonodictys muriformis) collected from a freshwater habitat in Thailand, which is morphologically similar to members of Monodictys S. Hughes . Neomonodictys is characterised by holoblastic, monoblastic, integrated, terminal, determinate conidiogenous cells and muriform, subglobose to globose, smooth-walled, pale brown to darkened to black conidia .
In this study, the fungus was isolated from submerged decaying wood in Erhai Lake, Yunnan Province in China. The morphology and phylogeny show that our collection is distinct from related species. We provide detailed descriptions, illustrations for Neomonodictys aquatica and a synopsis table for the morphology comparison.

Isolation and morphological examination
Submerged decaying wood was collected from Erhai Lake, Dali City, Yunnan, China. The coordinates of sampling sites are 25°44′29.65″N, 100°09′49.33″E and at an altitude of 1966 m. Samples were returned to the laboratory in plastic bags. The samples were incubated in aseptic plastic boxes, lined with moistened tissue paper at room temperature for one week. Specimen observations and morphological studies were conducted following the protocols provided by Luo et al. (2018b).
Morphological observations were made by using a SMZ760 series stereomicroscope and photographed using a Nikon-80i microscope. The fungal structures were measured with Tarosoft (R) Image Frame Work programme and images were processed using Adobe Photoshop CS6 extended version 13.0 (Adobe Systems, USA). Single spore isolation was carried out following the method described in Chomnunti et al. (2014). Germinating conidia were transferred aseptically to PDA plates with 0.5 mg/l of Amoxicillin and incubated at room temperature under dark conditions. The colonies were checked every three days. A herbarium was deposited in the herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (KUN-HKAS), Yunnan, China. Living cultures were deposited in Kunming Institute of Botany Culture Collection (KUNCC) and China General Microbiological Culture Collection Center (CGMCC). Facesoffungi numbers were registered as described in  and Index Fungorum numbers as in Index Fungorum(2021).

DNA Sequencing and Sequence Alignment
The appropriate fungal mycelium was scraped from the surface of colonies on Potato Dextrose Agar (PDA) plates with a scalpel into a 1.5 ml EP tube (Bao et al. 2018). Genomic DNA was extracted using the Trelief Plant Genomic DNA Kit (Beijing TsingKe Biological Technology and Services Co. Ltd, China) according to the manufacturer's protocols.
The primers ITS4/ITS5 for Internal transcribed spacer (ITS) and LR0R/LR5 for Large subunit ribosomal ribonucleic acid (LSU rRNA) were selected for PCR amplification (Vilgalys and Hester 1990). Polymerase Chain Reaction (PCR) mixture was performed in a 25 μl system reaction containing 9.5 μl ddH O, 12.5 μl of 2 × Power Taq PCR Master Mix, 1 μl of DNA template and 1 μl of each primer (10 μM) (Wang et al. 2019). The PCR thermal cycles for amplification of the ITS gene region were as per  and the LSU gene followed Sun et al. (2020). PCR amplifications were confirmed on 1% agarose electrophoresis gels stained with ethidium bromide.
Sequences were assembled with BioEdit. Sequences with high similarity indices were determined from a BLAST search to find the closest matches with taxa in Neomonodictys and from recently published data , Hyde et al. 2019). All consensus sequences and the reference sequences were aligned in MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/index.html, Katoh and Standley 2013). Aligned sequences of each gene region (ITS and LSU) were combined and manually improved using BioEdit v. 7.0.5.2 (Hall 1999). Ambiguous regions were excluded from the analyses and gaps were treated as missing data.

Phylogenetic Analyses
Maximum Likelihood analysis was performed in the CIPRES Science Gateway v.3.3 (Miller et al. 2010) using RAxML v. 8.2.8 as part of the "RAxML-HPC2 on XSEDE" tool (Stamatakis 2006, Stamatakis et al. 2008). The final ML search was conducted using the TM 2 GTRGAMMA + I model estimated using MrModeltest 2.2 (Nylander 2004), with ML bootstrap support being calculated from 1000 bootstrap replicates.
Bayesian analysis was performed using MrBayes v. 3.1.2. (Ronquist and Huelsenbeck 2003). The model of each gene was estimated using MrModeltest 2.2 (Nylander 2004), with GTR + I + G model being the best-fit model of ITS and LSU for Bayesian analysis. Posterior Probabilities (PP) (Rannala and Yang 1996) were performed by Markov Chain Monte Carlo sampling (MCMC) in MrBayes v.3.1.2 (Liu et al. 2012). Six simultaneous Markov chains were run for 50 million generations and trees were sampled every 5000 generation (resulting in 10,000 trees). The first 2000 trees, representing the burn-in phase of the analyses, were discarded and the remaining 8000 (post burning) trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree (Cai et al. 2006, Liu et al. 2012 Isolates and sequences used in this study (newly-generated sequences are indicated in bold and with "*" after species name, the type strains are in bold).

Phylogenetic analyses
The phylogram generated from Maximum Likelihood analysis, based on combined ITS and LSU sequence data, represents Pleurotheciales and the closely related orders. Seventynine strains are included in the combined analyses, which comprise 2039 characters (ITS: 849 bp, LSU: 1190 bp) after aligning. Leotia tubrica (AFTOL-1) is the outgroup taxon in this phylogentic tree. The best RAxML tree with a final likelihood value of -12803.740107 is presented. The matrix had 698 distinct alignment patterns with 34.21% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.222096, C =0.295691, G = 0.272214, T = 0.209999; substitution rates AC = 1.588217, AG = 2.820721, AT = 2.535737, CG = 1.003016, CT = 5.905028, GT = 1.000000; gamma distribution shape parameter α = 0.570011.
In the phylogenetic analysis, our new isolate Neomonodictys aquatica clustered as a sister taxon with N. muriformis with strong bootstrap support (99 ML/1.00 PP, Fig. 2).

Discussion
Up to now, two species are accepted in Neomonodictys, including the newly-introduced species. Both of them are collected from submerged wood in freshwater habitats  and only asexual morphs are reported. Morphologically, Neomonodictys is similar to Monodictys in having solitary, dictyospores conidia and monoblastic, hyaline to brown conidiogenous cells (Ellis 1971, Seifert et al. 2011. Compared with the diaphragms of them, Neomonodictys aquatica have a mostly transverse septum, less of the vertical septum, but the transerve and vertical septa of N. muriformis are evenly distributed. The significant difference between Neomonodictys and Monodictys is conidiophores, which are shorter than in the former (Kukwa and Diederich 2005). Phylogenetically, they are distinct ). In the phylogenetic analysis, Monodictys was placed in Dothideomycetes (Day et al. 2006, Seifert et al. 2011, Wijayawardene et al. 2020, while Neomonodictys was placed in Sordariomycetes ).

Figure 2.
Phylogenetic tree based on RAxML, generated from a combined ITS and LSU dataset.
Bootstrap support values for Maximum Likelihood (ML, black) higher than 75% and Bayesian posterior probabilities (BYPP, red) greater than 0.95 are indicated above the nodes as ML/PP. The tree is rooted to Leotia lubrica. The type-derived sequences are indicated in bold and new isolates are in red. Bootstrap values for Maximum Likelihood (ML) equal to or greater than 75% and clade credibility values greater than 0.90 from Bayesian-inference analysis labelled on the nodes. Ex-type strains are in bold and black, the new isolate is indicated in bold and red. (Fig. 2).