Taxonomic and Phylogenetic Characterizations Reveal Four New Species, Two New Asexual Morph Reports, and Six New Country Records of Bambusicolous Roussoella from China

During the ongoing investigation of bambusicolous ascomycetous fungi in Yunnan, China, 24 specimens belonging to the family Roussoellaceae were collected and identified based on morphological features and phylogenetic support. Maximum-likelihood (ML) analyses and Bayesian analyses were generated based on the combined data set of ITS, LSU, tef1, and rpb2 loci. The phylogenetic analyses revealed four novel lineages in Roussoella s. str.; thus, we introduced four new species viz., Roussoella multiloculate sp. nov., R. papillate sp. nov., R. sinensis sp. nov., and R. uniloculata sp. nov. Their morphological characters were compared with the known Roussoella taxa, which lack sequence data in the GenBank. Asexual morphs of R. kunmingensis and R. padinae were recorded from dead bamboo culms in China (from the natural substrates) for the first time. Neoroussoella bambusae, Roussoella japanensis, R. nitidula, R. padinae, R. scabrispora, and R. tuberculate were also reported as the first records from China. All new taxa are described and illustrated in detail. Plates are provided for new reports.

Roussoella (the type of genus of Roussoellaceae) was introduced by Saccardo and Paoletti [10] with R. nitidula Sacc. and Paol. as the type species recorded from bamboo Pure cultures were grown on PDA media, for 30-40 days, at 28 • C, in the dark. Fresh mycelium was scraped using a surgical knife and placed into a 1.5 mL centrifuge tube, and ground into powder using liquid nitrogen. Genomic DNA was extracted following the instruction book of the Biospin Fungus Genomic DNA Extraction Kit (BioFlux ® ).
Information of primers used for the amplification of internal transcribed spacers (ITS), small subunit rDNA (SSU), large subunit rDNA (LSU), translation elongation factor 1-α gene region (tef1), and RNA polymerase II second largest subunit (rpb2) genes is presented in Table 1. ITS, SSU, LSU, tef1, and rpb2 loci amplifications were performed by polymerase chain reaction (Eppendorf Mastercycler nexus gradient) according to the conditions presented in Table 2 [17]. Both forward and reverse primers (Table 1) were used for sequencing and these primers were the same as those used for amplification. The PCR products were sequenced, and the sequences were deposited in GenBank, as shown in Table 3. Table 1. ITS, SSU, LSU, tef1, and rpb2 loci primers information.
Bayesian analyses were performed using MrBayes v.3.0b4 [31]. MrModeltest v.2.2 selected the best evolution model [32]. Posterior probabilities (PP) [33,34] were performed by Markov Chain Monte Carlo sampling (MCMC) [35]. Six simultaneous Markov chains were run for 1,000,000 generations, and trees were sampled every 100th generation. The burn-in was set to 0.25, and the run was automatically stopped when the average standard deviation of split frequencies reached below 0.01 [36].
Trees were constructed (TreeView [37]) and formatted (Adobe Illustrator CS v.5). Maximum-likelihood bootstrap values (MLBP) equal to or greater than 50% and Bayesian posterior probabilities (BYPP) > 0.80 are given at the branches. The sequences used in this study are listed in Table 1. The alignment based on the combined loci and phylogenetic tree was submitted to TreeBASE under the code 29601 (https://www.treebase.org/, accessed on 20 April 2022).

Taxonomy
Culture characters: Ascospores and conidia germinating on PDA within 24 h and germ tubes produced from both cells and both sides. Notes: Roussoella kunmingensis is characterized by having immersed, uniloculate ascomata, cylindrical to cylindric-clavate, bitunicate asci, and ellipsoidal to fusiform, light brown to brown, 2-celled ascospores with longitudinal ribs. Multi-gene phylogenetic analyses showed that GMBCC1055, GMBCC1057, and GMBCC1086 grouped with R. kunmingensis (KUMCC 18-0128, ex-type) with high statistic values (100% MLBP, 1.00 BYPP). Base pair arrangement of ITS, tef1, and rpb2 regions of strains GMBCC1055, GMBCC1057, and GMBCC1086 and KUMCC 18-0128 were identical. However, Jiang et al. [5] described this species with only sexual morph from bamboo in China. Here, one new collection with sexual morph and two new collections with asexual morph were examined. Hence, we reported the asexual morph of R. kunmingensis for the first time and provide a description of the asexual morph and an illustration of the holomorph.
Culture characters: Ascospores germinating on PDA within 24 h and germ tubes produced from one cell. Colonies slow-growing, 20 mm diam. after 20 days at 28 • C, under 24 h dark, rounded, with even margin, white, cottony from above, and white at the margin, yellowish-brown at the center, ring-like from below.

Roussoella papillate
Culture characters: Conidia germinating on PDA within 24 h and germ tubes produced from one side. Colonies slow-growing, 40 mm diam. after 20 days at 28 • C, under 24 h dark, circular, with radialized margin, floccose at the center, brown from above and dark brown from below.
Distributions Notes: Phylogenetic analyses showed that the new strain GMBCC1126 grouped with R. padinae (MUT 5503, ex-type) which was introduced by Poli et al. [8] from brown alga Padina pavonica (i.e., from marine habitat). Base pair arrangement of ITS, LSU, and rpb2 loci are identical in strain GMBCC1126 and R. padinae. Therefore, GMB1320 (strain: GMBCC1126) and R. padinae are conspecific. However, sexual and asexual morphs of R. padinae are undetermined while Poli et al. [8] have described this species based only on the morphology of colonies and vegetative structures. In this study, we report the asexual morph of R. padinae for the first time from dead culms of bamboo. Further, this is the first record of this species from terrestrial habitats and the first record from China.  GMB1274, GMB1286 and GMB1279) are R. scabrispora. This is the first report of R. scabrispora from China.  Dai and Wijayaw. sp. nov. Figure 10. Index Fungorum number: IF 556014 Etymology: Reference to its first collection site China. Notes: Roussoella scabrispora comprises distinctive ascospores with reticulate wall ornamentation [1]. However, the ascospores of our new collection are slightly narrower (27-34 × 7-9.5 µm vs. (24−)25-29(−32) × (7−)9-10.5 µm) than in the protologue [39]. Nevertheless, based on phylogenetic analyses (Figure 1), we confirmed new collections (i.e., GMB1274, GMB1286 and GMB1279) are R. scabrispora. This is the first report of R. scabrispora from China.

Roussoella sinensis
Roussoella sinensis Dai and Wijayaw. sp. nov. Figure 10.  by large, immersed, eustromatic conidiomata, which are rather flattened, phialidic, annellidic conidiogenous cells and conidia covered by small tubercules [17]. Usually, the conidia of Roussoella species comprise verrucose wall ornamentation [1,39], while R. tuberculata has conidia with small and roughened tubercules. The above morphological identification is confirmed by our phylogenetic analyses and base-pair comparisons as well (100% MLBS, 1.00 BYPP, Figure 1). This is the first record of this species from China. while R. tuberculata has conidia with small and roughened tubercules. The above morphological identification is confirmed by our phylogenetic analyses and base-pair comparisons as well (100% MLBS, 1.00 BYPP, Figure 1). This is the first record of this species from China.   (Figure 1). Base pair differences of ITS and rpb2 gene loci of R. uniloculata and other related species showed that they are phylogenetically distinct species [38] (Table 6).   (Figure 1). Base pair differences of ITS and rpb2 gene loci of R. uniloculata and other related species showed that they are phylogenetically distinct species [38] ( Table 6). Roussoella uniloculata is similar to R. yunnanensis and R. siamensis in having ellipsoidal to fusiform and 2-celled ascospores with longitudinal striations. However, R. uniloculata can be distinguished from R. yunnanensis in having smaller ascostromata (250-350 µm diam. vs. 1-1.3 mm diam.) with single locule vs. with multiple locules, and asci with a short knob-like pedicel vs. asci with a slightly furcate short pedicel [5]. Roussoella uniloculata can be distinguished from R. siamensis in having smaller ascostromata (250-350 µm diam. vs. 620-750 µm diam. [17], higher locules (120-145 µm high vs. 70-120 µm high; [17]. Roussoella uniloculata can be compared with R. pustulans, in having small ascospores with bigger upper cells (8.5-12 × 3.5-4.5 µm vs. 10-16 × 4-5 µm) [1]. However, R. uniloculata differs by smaller ascostromata (165-300 µm diam. vs. 1 mm diam.), and in the phylogenetic tree, they form separate lineages (Figure 1). Moreover, morphology, host, and distribution of the new species were compared with the known species which are lacking sequence data (Table 7). Hence, based on molecular phylogenetic and morphological analyses, we introduced R. uniloculata as a novel species of Roussoella. Table 7. Ascospores, host, and distribution comparison of eleven sequence lacking known Roussoella taxa with three new species in this study. "-": not available in the protologue.
However, asexual morphs of R. kunmingensis (described initially as a sexual taxon [8]) and R. padinae (described initially as without sexual or asexual characteristics fide [8]) were reported for the first time in the present study. Sexual and asexual links were established based on DNA sequence analyses (Figure 1). The sexual morph of R. kunmingensis was also reported from Kunming, Yunnan Province. Nevertheless, Poli et al. [8] introduced R. padinae