Synopsis of Leptosphaeriaceae and Introduction of Three New Taxa and One New Record from China

Leptosphaeriaceae, a diverse family in the order Pleosporales, is remarkable for its scleroplectenchymatous or plectenchymatous peridium cells. Four Leptosphaeriaceae species were discovered and studied during the investigation of saprobic fungi from plant substrates in China. Novel taxa were defined using multiloci phylogenetic analyses and are supported by morphology. Based on maximum likelihood (ML) and Bayesian inference (BI) analyses, these isolates represent three novel taxa and one new record within Leptosphaeriaceae. A new genus, Angularia, is introduced to accommodate Angularia xanthoceratis, with a synopsis chart for 15 genera in Leptosphaeriaceae. This study also revealed a new species, Plenodomus changchunensis, and a new record of Alternariaster centaureae-diffusae. These species add to the increasing number of fungi known from China.

The GTR + GAMMA model of nucleotide evolution was used for the datasets, and RAxML rapid bootstrapping of 1000 replicates was performed. The best-fit evolutionary models for individual and combined datasets were estimated under the Akaike Information Criterion (AIC) using jModeltest 2.1.10 on the CIPRES web portal for posterior probability [48]. The GTR model was the best model for all the datasets. Bayesian inference analyses were performed using MrBayes v. 3.2.6 on the CIPRES web portal [49]. Simultaneous Markov chains were run for seven million generations, and trees were sampled every 100th generations.
FigTree v. 1.4 [50] was used to visualize phylogenetic trees. The phylogram was edited by using Adobe Illustrator CS v. 6. All newly generated sequences were deposited in GenBank. All the alignments and trees were deposited in TreeBASE (Submission ID: 29394 and 29395).

Taxonomy
Culture characteristics: Colonies on PDA reaching 30 mm diam. after 3 weeks at 25 • C. Cultures from above, gray in the center, milky white radiating outward, dense, circular, creeping hyphae, grayish-green at the margins; reverse dark at the center, milky white radiating outward. Yellow pigmentation diffused into the media.
GenBank accession numbers: LSU = OL897174, SSU = OL984031, ITS = OL996123, and tub2 = OM009247 Notes: Plenodomus changchunensis (CCMJ5011 and CCMJ5012) formed a sister clade distinct from Plenodomus lindquistii with 99% ML/1.00 BPP support based on phylogenetic analysis of the concatenated ITS, LSU, SSU, and tub2 datasets (Figure 2). Plenodomus changchunensis is similar to P. lindquistii in the size of conidia [51]. This species can be distinguished from P. lindquistii (CBS 381.67) by 34 nucleotides in the ITS region (34/643 in the ITS region and 0/866 in the LSU region). In the BLASTn search, the closest match to the LSU and ITS sequences of P. changchunensis were 100% and 89.57% similar to Leptosphaeria sp. (PHY-30) and P. lindquistii (MCN535002) with 95% query cover which translates to a 95% and 85.1% similarity, respectively. Plenodomus changchunensis was found associated with a grass near the water resources in temperate regions. Therefore, this fungus is introduced as a novel species. Notes: Alternariaster centaureae-diffusae was originally described from the dead stems of Centaurea diffusa Lam. in Russia [4]. The new isolate (HMJAU 60188) has similar morphology to the type strain of A. centaureae-diffusae (MFLU 15-1521) in having fasciculate, filiform, constricted at the apical septum, conical, yellowish-brown ascospores with swollen apical cell [4]. A pairwise comparison of the sequences of the new isolate (HMJAU 60188) with the type species of A. centaureae-diffusae revealed minor differences. The new isolate clustered in the same clade as the type strain of A. centaureae-diffusae ( Figure 2). Therefore, we report A. centaureae-diffusae on Clematis spp. as a new host and new geological record.
isolate clustered in the same clade as the type strain of A. centaureae-diffusae ( Figure 2). Therefore, we report A. centaureae-diffusae on Clematis spp. as a new host and new geological record.

Discussion
Molecular biology has helped to elucidate the phylogenetic relationships among members of Dothideomycetes, particularly among several phoma-like taxa [19,48]. Multiloci analyses based on LSU, SSU, ITS, tub2, rpb2, and tef-1 sequences have been widely used to define species boundaries in Leptosphaeriaceae and other families of Dothideomycetes [19,48,49]. We carried out phylogenetic analyses with a concatenated dataset of five loci

Discussion
Molecular biology has helped to elucidate the phylogenetic relationships among members of Dothideomycetes, particularly among several phoma-like taxa [13,52]. Multi-loci analyses based on LSU, SSU, ITS, tub2, rpb2, and tef -1 sequences have been widely used to define species boundaries in Leptosphaeriaceae and other families of Dothideomycetes [13,52,53]. We carried out phylogenetic analyses with a concatenated dataset of five loci (ITS, LSU, SSU, tub2, and rbp2) for Leptosphaeriaceae members. The final alignment included 138 strains representing 132 ingroup taxa and six outgroup strains. However, the Plenodomus species were polyphyletic and mixed with Alternariaster, Ochraceocephala, and Praeclarispora taxa. It is often encouraged to use additional taxon-specific secondary barcode loci to delineate taxa.
We therefore compared the phylogenetic informativeness of tub2 (52 sequences translated to 37.7%) and rpb2 (46 sequences translated to 33.3%) sequences of Leptosphaeriaceae. Our study shows that the polyphyletic topology of the Plenodumus group is due to the rpb2 gene ( Figures S2-S4). This could be due to a lack of rpb2 barcodes in several related taxa, but the rpb2 gene can be useful for delineation at the genus level [12,41]. In contrast, using the tub2 gene provides a better resolution at the species level within the genera (Figure 2). Therefore, we performed phylogenetic analyses of Leptosphaeriaceae species with a concatenated dataset of ITS, LSU, SSU, and tub2 loci. Three new species of Leptosphaeriaceae were revealed from China based on multilocus phylogeny combined with morphology.
The phylogeny from our analyses is similar to several previous studies [4,12,13]. The Leptosphaeriaceae taxa clustered in fifteen clades based on the ITS, LSU, SSU, and tub2 datasets. A novel genus Angularia is also introduced in Leptosphaeriaceae to accommodate a new species, A. xanthoceratis. Conidial characteristics are the primary morphological characteristics that distinguish Angularia from the allied genus Sphaerellopsis (Figure 1). Plenodomus formed a separate clade, sister to Ochraceocephala, and revealed a novel species P. changchunensis with strong support. Many new genera have been introduced in Leptosphaeriaceae [2,4,8,[12][13][14]23], which indicates that this family has a high degree of fungal diversity and distribution.
Plenodomus lingam was chosen to be the representative type species of Plenodomus over P. rabenhorstii Preuss [14,54]. There are 36 epithets listed under Plenodomus in Species Fungorum (2022) and 107 epithets in MycoBank. The host specificity of Plenodomus has not yet been clarified as species have been recorded from various plant families (Asteraceae, Fabaceae, Lamiaceae, and Liliaceae) [9]. In our study, P. changchunensis was found on Poaceae, which suggests that the Leptosphaeriaceae species are widely associated with many types of substrates. Members of Plenodomus appear to be cosmopolitan, as they have been recorded in both temperate and tropical countries (China, Greece, France, Japan, Netherlands, Peru, and Spain) [55].
Alternariaster centaureae-diffusae has been isolated from Centaurea diffusa Lam. (Asteraceae) in Shakhty city, Rostov region, Russia [4]. In this study, it was isolated from Clematis spp. (Ranunculaceae) in Kunming, Yunnan province, China. Therefore, our study extended the host range of A. centaureae-diffusae even though the environment of the two cities is different (temperate and subtropical). Therefore, we speculate that this species could be found in different environments and hosts [56].
Fungal diversity and taxonomy are constantly changing, necessitating a continuous assessment [57][58][59]. It is especially significant where taxa are described from genera that usually accommodate pathogens [60,61]. For example, Plenodomus and Alternariaster are the causal agents of blackleg disease and leaf spots of Helianthus annuus (sunflower) worldwide [31,32,62,63]. The discovery of novel species in a pathogenic genus could also indicate the discovery of emerging pathogens that can cause damage to economically important crops [64,65]. The formation of new fungi species has been reported to be intricately linked to their evolutionary relationships and ecological roles [20]. These phenomena can also occur when species are associated with different hosts and environments, as in the case of A. centaureae-diffusae in this study. The presence of the Alternariaster and Plenodomus species in different substrates reflects their ecological importance. Further studies focusing on fungal diversity from different niches are needed to understand the relationships between these organisms in ecosystems.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/jof8050416/s1, Figure S1: Phylogram generated from Bayesian inference analysis based on combined ITS, LSU, SSU, and tub2 sequence data. Figure S2: Phylogram generated from maximum likelihood analysis based on combined ITS, LSU, SSU, tub2, and rpb2 sequence data. Figure S3: Phylogram generated from maximum likelihood analysis based on combined ITS, LSU, SSU, and rpb2 sequence data. Figure S4: Phylogram generated from maximum likelihood analysis using rpb2 sequence data. Figure S5: Phylogram generated from maximum likelihood analysis using tub2 sequence data.