Sporidesmiellalignicola sp. nov., a new hyphomycetous fungus from freshwater habitats in China

Abstract Background Freshwater fungi, growing on submerged wood, can promote the degradation of organisms and the reuse of rotten wood energy and play key roles in freshwater ecosystems. Here, a new hyphomycetous fungus, Sporidesmiellalignicola, was isolated and identified from submerged wood samples collected in a small stream in Jiangxi Province, south-eastern China. New information The new taxon was studied, based on morphological characters and phylogenetic analyses combined with LSU, ITS, TEF1α and RPB2 sequences data. Sporidesmiellalignicola was morphologically characterised by its pink colonies on PDA medium in prophase, macronematous, mononematous, solitary, brown, septate, unbranched, straight or slightly flexuous conidiophores with holoblastic, polyblastic, integrated, terminal, pale brown conidiogenous cells and cylindrical, narrowly clavate, broadly obovoid to cuneiform, 3–4-distoseptate, olivaceous brown or brown conidia with rounded apex. A phylogenetic tree was constructed, based on the combination of LSU, ITS, TEF1α and RPB2 sequences data.

So far, the molecular data of Sporidesmiella are relatively few; there are DNA sequences of only five species deposited in NCBI, i.e. S. aquatic, S. fusiformis, S. hyalosperma, S. novae-zelandiae and S. obovoidia. Therefore, most Sporidesmiella species have not been subjected to molecular phylogenetic analysis. Shenoy et al. (2006) classified Sporidesmiella fusiformis in the Melanommataceae according to the phylogenies with the combined LSU nu-rDNA and RPB2 dataset. Luo et al. (2019), Crous et al. (2020 and Dong et al. (2021) accommodated S. aquatic, S. hyalosperma, S. novae-zelandiae and S. obovoidia within Junewangiaceae, based on the combination of LSU, ITS, TEF1α and RPB2 sequences data. Therefore, as Sporidesmiella was suspected to be polyphyletic, the molecular data of the type species S. claviformis are in need of analysis.
Based on investigations of freshwater fungi in Jiangxi Province (Hu et al. 2012a, Huang et al. 2016, Song et al. 2018, Song et al. 2020, we reported a new species of Sporidesmiella, collected on submerged wood from freshwater habitats in Jiangxi Province. It was described and illustrated as Sporidesmiella lignicola, based on phylogenetic evidence of combined LSU, ITS, TEF1α and RPB2 sequence data and morphological characters.

Samples collection
Submerged wood samples were collected randomly from a stream in Xinfeng County, Ganzhou City, Jiangxi Province, China. The samples were taken to the laboratory in ziplock bags and incubated in moist plastic boxes.

Specimen examination
Fruiting bodies or colonies were examined following the method of Hu et al. (2012a) using a Nikon dissecting microscope. Samples were examined and photographed using a Nikon (Ni) compound microscope with differential interference contrast (DIC) . The fungal specimens were deposited in the Herbarium of Fungi, Jiangxi Agricultural University (HFJAU), Nanchang, China.

Single spore isolation and cultivation
The fungal colonies on the rotten wood were picked up and placed in 200 μl sterile water to make a suspension, then the suspension was evenly spotted on potato dextrose agar (PDA), then cultured in a 28℃ incubator. The spore germination was observed every 12 hours and recorded. The germinating single spore was transfered to new PDA medium with a sterile needle under aseptic conditions and then cultured in a 28℃ incubator to obtain the pure strain.

DNA extraction, PCR amplification and sequencing
DNA was extracted from the pure cultures with the CTAB method, following Doyle and Doyle (1987). Four gene regions, LSU, ITS, TEF1α and RPB2 were amplified using the primer pairs LR0R/LR5, ITS1/ITS4, EF1-983F/EF1-2218R and RPB2-5F/RPB2-7cR, respectively (Vilgalys and Hester 1990, White et al. 1990, Liu et al. 1999). The amplification was performed following the method described by Hu et al. (2012b). The PCR products were examined using 1% agarose electrophoresis gels, stained with GelRed and purified and sequenced with the same primers at Tsingke Biotechnology Co. Ltd.
The ML analyses were conducted with RAxML v. 7.2.6 (Stamatakis and Alachiotis 2010), using a GTRGAMMA substitution model with 1000 bootstrap replicates. The robustness of the analyses was evaluated by bootstrap support (MLBS).    broadly obovoid to cuneiform, truncate at the base, rounded or rarely coronate at the apex, 2-3-distoseptate, pale olivaceous to olivaceous brown or brown, smooth. Conidial session schizolytic (Fig. 1).

Ecology
Saprophyte on wood submerged in a small stream.

Notes
Sporidesmiella lignicola is characterised by being cylindrical, broadly obovoid to cuneiform, truncate at the base, rounded at the apex, 3-4-distoseptate, pale olivaceous brown to brown, smooth, which is consistent with the characteristics of Sporidesmiella. Sporidesmiella lignicola is similar to S. obovoidia and S. hyalosperma in having polyblastic conidiogenous cells and obovoid, 3-4-distoseptate, brown conidia (Luo et al. 2019, Dong et al. 2021). However, S. lignicola differs from other species in having longer and verrucose conidiophores (Table 2). In addition, the colonies of S. lignicola are pink from above, pink-grey from below, characteristics which were not observed in the other two species (Fig. 3, Table 2). Based on a BLAST of NCBI's GenBank nucleotide database, the most similar sequence was Sporidesmiella obovoidia. The nucleotide comparison between S. lignicola and S. obovoidia showed differences of 10 and 4 nucleotides in ITS and LSU sequence data, respectively (Fig. 4, Fig. 5), which supported them to be different species (Jeewon and Hyde 2016).
Unfortunately, the strain could not be successfully activated due to improper operation during preservation. When the original culture was retained for 6 months, the Table 2.
Comparisons of Sporidesmiella lignicola and similar species. The specific base differences between S. lignicola and S. obovoidia in ITS. Different base pairs have been marked on specific sites, and the same base is omitted.
sporulation of mycelium could be observed under the microscope (Fig. 2). We deposited the dried culture as specimens (HFJAU 10001) of this species. The specific base differences between S. lignicola and S. obovoidia in LSU. Different base pairs have been marked on specific sites, and the same base is omitted.

Phylogenetic analyses
The analysed dataset comprised 35 taxa retrieved from GenBank and we selected Botryotinia fuckeliana (AFTOL-ID 59) as the outgroup taxon (Table 1). Partial nucleotide sequences of LSU (844bp), ITS (598bp), TEF1α (881bp), RPB2 (1059bp) and, for a total of 3382 characters including gaps, were used to determine the phylogenetic placement of the new taxon. The generated ML and Bayesian trees were similar in topology and the best scoring RAxML tree is presented in Fig. 6.
The phylogenetic tree demonstrated that the new taxon (Sporidesmiella lignicola), together with species of S. obovoidia, S. hyalosperma, S. aquatica and S. novae-zelandiae, formed a distinct clade representing the genus Sporidesmiella with strong bootstrap support (100% MLBS, 1.00 PP). Additionally, in our phylogenetic analysis, the three genera Dictyosporella, Junewangia and Sporidesmiella constituted a well-supported clade with strong ML and BYPP bootstrap support (100% MLBS, 1.00 PP), which is in accordance with Luo et al.

Discussion
Many species of Sporidesmiella are found on decaying leaves, wood, bark, dead branches, cane and culms. At present, only three species have been found on submerged wood. Our research provides a new freshwater fungus found on submerged wood for Sporidesmiella and we provide four new sequences data, enriching the molecular database of Sporidesmiella.
As a decomposer, lignicolous freshwater fungi play an important role in freshwater ecosystem and material cycles in nature. They are also important biological resources, which have great application potential. Lignicolous freshwater fungi are a great treasure of resources to be developed. Many unknown species are waiting for us to understand and explore.