New Species of Didymellaceae within Aquatic Plants from Southwestern China

Members of Didymellaceae have a wide geographical distribution throughout different ecosystems, and most species are associated with fruit, leaf, stem and root diseases of land plants. However, species that occur in aquatic plants are not clearly known. During a survey of the diversity of endophytes in aquatic plants in Yunnan, Sichuan, and Guizhou provinces, we obtained 51 isolates belonging to Didymellaceae based on internal transcribed spacer region (ITS) sequences. Further, the phylogenetic positions of these isolates were determined by combined sequences composed of ITS, partial large subunit nrRNA gene (28S nrDNA; LSU), RNA polymerase II second largest subunit (rpb2) and partial beta-tubulin gene (tub2). Combining morphological characteristics and multi-locus phylogenetic analyses, two new varieties belong to Boeremia and 12 new species distributed into seven genera were recognized from 51 isolates, i.e., Cumuliphoma, Didymella, Dimorphoma, Ectophoma, Leptosphaerulina, Remotididymella, and Stagonosporopsis. Among these species, only one species of Stagonosporopsis and two species of Leptosphaerulina show teleomorphic stages on OA, but have no anamorphic state. Each new species is described in detail, and the differences between new species and their phylogenetically related species are discussed here. The high frequency of new species indicates that aquatic plants may be a special ecological niche which highly promotes species differentiation. At the same time, the frequent occurrence of new species may indicate the need for extensive investigation of fungal resources in those aquatic environments where fungal diversity may be underestimated.


Introduction
Aquatic plants are the plant groups that remain submerged or float on the surface of the water [1]. Based on the different habitats and physiological characteristics during their life cycle, aquatic plants are generally classified into five types: emergent plants, floating-leaved plants, free-floating plants, submerged plants, and wet plants [2,3]. Most aquatic plants are well adapted to their surroundings, and they play an important role in maintaining the normal functioning of aquatic ecosystems [4]. Aquatic plants play an important role in maintaining water quality, including removing excess nutrient loads, absorbing nutrient mineral ions and reducing sediment resuspension [5,6].
Fungal endophytes refer to the kinds of fungi that live within plant tissues (roots, stems and leaves, etc.) without causing disease in plants [7]. They play an active role in plant growth by protecting the host plant from pests and pathogens and improving the adaptability of plants to extreme environments [8][9][10]. In addition, many studies have reported that fungal endophytes frequently produce diverse active metabolites that contribute to the well-being of the host plant [11]. Some endophytes can be symbiotic or beneficial in one case, such as providing resistance to hosts against different biotic and abiotic stresses, but potential pathogens in another, such as the barley pathogen publication [41]. At each sampling site, dominant plants were collected. For each species, at least 15 individuals were collected, with at least 5-10 m between any two individuals. Healthy and mature plants with undamaged leaves were completely uprooted and cleaned, then complete plant samples were transported back to the laboratory and stored at 4 • C.

Isolation of Endophytic Fungi
After the aquatic plant samples were thoroughly washed with tap water, each plant sample was cut into 20-30 mm, and subsequently, the plant samples were processed in the following order: First, the samples were placed in sterile distilled water (30 s), then immersed in 0.5% sodium hypochlorite (2 min), then in sterile distilled water (1 min), then in 75% ethanol (2 min), and finally again in sterile distilled water (1 min) [42]. After surface sterilization, plant tissues (roots, stems and leaves) were cut into small pieces of approximately 5 mm length and placed uniformly in Petri dishes containing Rose Bengal Agar (RBA; Guangdong Huan Kai Microbial Technology Co., Ltd., Guangzhou, China). Samples were incubated at 25 • C and observed for growth periodically. When the colonies grew to a level suitable for isolation and identification, the mycelium was picked onto a new plate containing potato dextrose agar (PDA; 200 g potato, 20 g dextrose, 18 g agar, 1000 mL distilled water) for purification.
The pure culture and dried culture specimens of this study were stored in the Herbarium of the Laboratory for Conservation and Utilization of Bio-Resources, Yunnan University, Kunming, Yunnan, China (YMF).

Morphological Characterization
Agar plugs removed from fresh culture on PDA were incubated on oatmeal agar (OA; 40 g oatmeal, 18 g agar, 1000 mL distilled water), malt extract agar (MEA; 30 g malt powder, 3 g peptone, 18 g agar, 1000 mL distilled water) and PDA at 25 • C to induce sporulation. Colony diameter and culture traits were determined at 7 days and 14 days of incubation, respectively. Colony colors were assessed according to the charts of Rayner [43]. Micromorphological structures of mature ascomata/conidiomata, ascospores/conidia and conidiogenous cells from OA cultures were placed in sterile distilled water for observation. Observation was performed using a light microscope (Olympus BX51, Japan) under differential interference contrast (DIC) illumination, using an Olympus DP controller (v.3,1,1208) software of the Olympus DP 10 digital camera to capture. The average measurements of the structures were calculated from 30 measurements.
All PCR products were tested by 1.5% agarose gel electrophoresis, and then purified using a commercial kit (Bioteke Biotechnology, Wuxi, China) according to the manufacturer's instructions. The purified PCR products were sequenced forward and reverse using a LI-COR 4000 L automated sequencer and Thermo Sequenase-kit as described by Kindermann et al. [52]. Raw sequence chromatograms were checked manually, and then each isolate was compared using ClustalW in MEGA 6.06 to generate concordant sequences [53]. The consensus sequences were deposited in the GenBank database at the National Center for Biotechnology Information (NCBI). The reference sequences and new sequences generated in this study are listed in Supplementary Table S1.

Sequence Alignment and Phylogenetic Analysis
The phylogenetic tree was constructed with four loci, ITS, LSU, rpb2 and tub2, to identify these isolates of Didymellaceae at the species level. The reference strains were selected on the basis of the high sequence similarity of BLAST searches of ITS in GenBank and the adjacent strains provided by recent studies of Didymellaceae [54][55][56][57][58][59]. We selected 310 isolates that had been sequenced and deposited in GenBank as reference strains, referring to 34 representative genera. All sequences used in this study are listed in Supplementary Table  S1. The generated sequences were manually aligned with CLUSTAL_X v. 1.83 [60] with default parameters. Aligned sequences of multiple loci were concatenated and manually adjusted through BioEdit v. 7.0.4.1 [61], and ambiguously aligned regions were excluded. The resulting FASTA file contains 2417 characters with gaps; ITS contains 482 sites, LSU contains 968 sites, rpb2 contains 612 sites, and tub2 contains 355 sites. Bayesian inference (BI) and maximum-likelihood (ML) methods were used for phylogenetic analyses of the final FASTA file.
For ML analysis, the built-in file ModelFinder in IQ-TREE was invoked to determine the best model for concatenating genomic FASTA files [62]. TIM2e+I+G4 was used for the final ML search, and maximum-likelihood bootstrap support values (MLBP) were calculated with 1000 replicates.
For BI analysis, the resulting FASTA file was converted to a NEXUS file using MEGA7 [63]. The NEXUS file was executed with MrBayes v. 3.2.2 [64]. The Akaike information criterion (AIC) implemented in jModelTest version 2.0 was used to select the best fit models after likelihood score calculations [65]. HKY+I+G was estimated as the best-fit model under the output strategy of AIC, Lsetnst = 6, rates = gamma. A Markov Chain Monte Carlo (MCMC) algorithm was used to generate phylogenetic trees with Bayesian probabilities. Two runs were executed simultaneously for 6,000,000 generations and sampled every 1000th generation; four chains containing one cold and three heated were run until the average standard deviation of the split frequencies dropped below 0.01, and the stationarity of the analyses was confirmed in line with the standards described by Sun and Guo [66]. The initial 25% of the generations of MCMC sampling were discarded as burn-in. The refinement of the phylogenetic tree was used for estimating BI posterior probability (BIPP) values. FigureTree v1.4.3 was used to visualize the phylogenetic The Bayesian posterior probabilities (BIPP) above 0.80 and RAxML bootstrap support values (MLBS) above 70% are given at the nodes (BIPP/MLBS). Some of the basal branches were shortened to facilitate layout. Genera are delimited in colored boxes, with the genus name indicated to the right. Strains with special status are indicated with a superscript letter after the accession number (R: representative; T: ex-type). The new species obtained in this research are printed in bold. The tree is rooted to Coniothyrium palmarum culture CBS 400.71.

Phylogenetic Analysis
All Didymellaceae spp. isolates were first identified at the family level based on their ITS sequences. To further identify these isolates at the genus and species level, we conducted four-locus (ITS, LSU, rpb2, and tub2) phylogenetic analysis referring to 2417 characters and 362 sequences, 310 reference sequences registered from GenBank and 51 representative sequences of Didymellaceae isolates obtained in this study. Coniothyrium palmarum culture CBS 400.71 served as outgroup. The four individual sequence datasets did not show conflicts in the tree topologies based on preliminary ML analyses, which allowed us to combine the four genes for the multi-locus analysis. The topological structure of the phylogenetic tree constructed by ML and BI was congruent; only the Bayesian tree is shown in this study ( Figure 1).
The phylogenetic tree showed that 51 isolates were distributed in 10 genera, including 12 new species, two new varieties and 14 known species. Fifteen isolates fell into the genus Boeremia; of these, four isolates clustered with Boeremia inicola (BIPP 0.9/MLBS 100), and we designated these isolates as Boeremia exigua var. vulgaris. Seven isolates were proposed to be new varieties Boeremia exigua var.

Taxonomy
Boeremia exigua var. vulgaris Y. Huang and Z.F. Yu, sp. nov. (Figure 2).  Etymology: Named after the host species from which the holotype was collected, isolated as an endophyte from the stem of Hippuris vulgaris L.
Culture characteristics: Colonies on OA, 38-40 mm diam after 7 days, margin irregular, covered by dark brown aerial mycelia, flat, white near the margin; reverse concolorous. Colonies on PDA, 22-28 mm diam after 7 days, margin irregular, aerial mycelia dark brown, white near the margin; reverse brown, white near the margin. Colonies on MEA, 40-42 mm diam after 7 days, margin irregular, brown, white near the margin; reverse brown, white near the margin. Application of NaOH results in a brown to blackish green discoloration.
Additional  Notes: Based on multi-locus phylogenetic analysis, seven Boeremia exigua var. kasaensis isolates formed a solitary clade and near B. exigua var. pseudolilacis Aveskamp, Gruyter and Verkley. It is distinguished from B. exigua var. pseudolilacis by the discoloration after application of NaOH to the culture (blackish green discoloration on B. exigua var. kasaensis, and no effect on B. exigua var. pseudolilacis), and the conidial matrix is cream instead of rosy-buff [70].
Culture characteristics: Colonies on OA, 70-75 mm diam after 7 days, margin regular, grayish black, covered by white floccose aerial mycelia; reverse black to grayish white. Colonies on PDA, 80-82 mm diam after 7 days, margin regular, densely covered by white floccose aerial mycelia; reverse white, with concentric circles of grayish white to yellowish. Colonies on MEA, 76-80 mm diam after 7 days, margin regular, white to pale yellow, covered by white floccose aerial mycelia; reverse concolorous. NaOH test negative.
Culture characteristics: Colonies on OA, 68-70 mm diam after 7 days, margin regular, covered by greenish olivaceous aerial mycelia, dense, with concentric circles of pale olivaceous, smoke gray near the center; reverse olivaceous to white. Colonies on PDA, 65-69 mm diam after 7 days, margin regular, aerial mycelia woolly, dark gray to white, forming some radial lines near the center; reverse gray to white. Colonies on MEA, 59-68 mm diam after 7 days, margin regular, olivaceous, with sparse white aerial mycelia near the center; reverse concolorous. Application of NaOH results in a reddish brown discoloration of the agar.
Culture characteristics: Colonies on OA, 64-68 mm diam after 7 days, margin regular, dark brown, covered by floccose aerial mycelia, whitish; reverse brown. Colonies on PDA, 69-70 mm diam after 7 days, margin regular, aerial mycelia woolly, grayish white; reverse brown, white near the margin. Colonies on MEA, 65-67 mm diam after 7 days, margin regular, covered by floccose aerial mycelia, dense, pale brown to white; reverse dark brown to white. NaOH test negative.
Additional Notes: Based on multi-locus phylogenetic analysis, four strains of Didymella hippuris formed a solitary clade, and phylogenetically near Did. Pomorum Qian Chen and L. Cai, but it can be distinguished by the conidial matrix being brown rather than cream white [72]. Furthermore, the conidia of Did. hippuris are wider than those of Did. pomorum (2.5-3.6 µm vs. 1.5-2.5(-3) µm) [73].
Culture characteristics: Colonies on OA, 60-67 mm diam after 7 days, margin regular, grayish brown, covered by flat and gray aerial mycelia; reverse brown. Colonies on PDA, 60-70 mm diam after 7 days, margin regular, grayish brown; reverse dark brown. Colonies on MEA, 58-66 mm diam after 7 days, margin irregular, dark brown, covered by brown aerial mycelia, some radially furrowed zones near the center; reverse dark brown, white near the margin. Application of NaOH results in a brown discoloration of the agar.
Additional specimen examined: China, Yunnan province, Dali Bai Autonomous Prefecture, Dali city, Erhai Lake, isolated as an endophyte from the root of  Etymology: Named after the host genus Hippuris, from which the holotype was isolated.
Didymella myriophyllana Y. Huang and Z.F. Yu, sp. nov. (Figure 8). Etymology: Named after the host species from which the holotype was collected, isolated as an endophyte from the stem of Myriophyllum aquaticum.
Culture characteristics: Colonies on OA, 50-55 mm diam after 7 days, margin regular, covered by flat aerial mycelia, brown to pale yellow; reverse brown, pale yellow near the margin. Colonies on PDA, 45-50 mm diam after 7 days, margin irregular, grayish brown, covered by floccose aerial mycelia; reverse black, grayish white near the margin. Colonies on MEA, 40-45 mm diam after 7 days, margin irregular, covered by floccose aerial mycelia, blackish green to dark brown, pale yellow near the margin; reverse black, white near the margin. NaOH test negative. Etymology: Named after the host species from which the holotype was collected, isolated as an endophyte from the stem of Myriophyllum aquaticum.
Holotype: China, Sichuan province, Ganzi Tibetan Autonomous Prefecture, Luhuo county, The Kasa Lake Nature Reserve, isolated as an endophyte from the leaf of Myriophyllum aquaticum, September 2016, Y. Huang. Holotype culture YMF1.05100. Note: Based on multi-locus phylogenetic analysis, two strains of Didymella myriophyllana formed a solitary clade, and near a new species Did. gongkasis. Morphologically, the conidia of Did. myriophyllana are narrower than those of Did. gongkasis, 1.6-2.5 µm vs. 2-2.8 µm. Moreover, this species is distinguishable from Did. gongkasis by the absence of a positive reaction to NaOH.
Leptosphaerulina shangrilensis Y. Huang and Z.F. Yu, sp. nov. (Figure 9). Culture characteristics: Colonies on OA, 33-35 mm diam after 7 days, margin regular, grayish black, covered by grayish white aerial mycelia, sparse; reverse black. Colonies on PDA, 38-45 mm diam after 7 days, margin regular, dark brown with some gray section, covered by floccose aerial mycelia; reverse black, white near the margin. Colonies on MEA, 40-41 mm diam after 7 days, margin regular, dark brown with some grayish white section, covered by floccose aerial mycelia; reverse black. Application of NaOH results in a brown to reddish brown discoloration.
Culture characteristics: Colonies on OA, 50-56 mm diam after 7 days, margin regular, brown with some grayish white section, covered by floccose aerial mycelia; reverse dark brown to pale brown. Colonies on PDA, 53-55 mm diam after 7 days, margin regular, pale brown with some grayish white section, covered by floccose aerial mycelia; reverse brown, grayish white near the margin. Colonies on MEA, 40-42 mm diam after 7 days, margin regular, brown, covered by floccose aerial mycelia, white near the margin; reverse concolorous. Application of NaOH results in a blackish green to dark brown discoloration.
Note: In the phylogenetic tree Cumuliphoma lijiangensis, which was isolated from Lijiang city, formed an independent lineage basal to the genus Cumuliphoma, and C. lijiangensis  (Figure 1). This species is morphologically closely related to C. omnivirens. However, C. lijiangensis does not produce chlamydospores [78]. Also, ITS sequence comparison revealed 3 base pair differences between C. lijiangensis and C. indica, and 4 base pair differences between C. lijiangensis and C. pneumoniae [78,79].
Culture characteristics: Colonies on OA, 55-60 mm diam after 7 days, margin regular, covered by flat aerial mycelia, dark olivaceous, gray near the center; reverse concolorous. Colonies on PDA, 50-55 mm diam after 7 days, margin irregular, aerial mycelia greenish olivaceous, white near the margin; reverse concentric circles of different color, dark olivaceous near the center, dark brown to pale yellow. Colonies on MEA, 55-60 mm diam after 7 days, margin regular, yellowish brown, white near the center; reverse brown, white near the margin. Application of NaOH results in a brown to reddish discoloration.

Discussion
In recent years, the vast majority of studies on the diversity of Didymellaceae have focused on the soil environment [23,25,36,78,81,82]. In contrast, the diversity of this family in freshwater ecosystems has received very little attention. In our study, we investigated the species diversity of the Didymellaceae fungi of aquatic plants in southwestern China, and the sampling sites included wetlands, lakes and ponds in Yunnan, Sichuan and Guizhou provinces. Among 51 Didymellaceae strains, 33 isolates represented 12 new species and two new varieties, and high frequency occurrence of new species showed that aquatic plants may be a special ecological niche that highly promotes species differentiation. New species were distributed in 10 genera, mainly in Boeremia, Didymella, Epicoccum, Leptosphaerulina and Stagonosporopsis, and these dominant genera are also frequently reported in terrestrial ecosystems [23,70].
Didymella was the most abundant genus in the study and was isolated from seven aquatic plants collected from 12 sampling sites. Previously, Didymella was reported as a worldwide fungus that often takes advantage of specific conditions to colonize plants, occasionally causing serious damage, such as stem cankers and black spot disease of fruits and leaves [83,84]. At the same time, Didymella is the only saprophytic genus that is related to Ascochyta and Phoma [22,27,85], and the question of the polyphyly of Ascochyta, Didymella and Phoma remains unresolved [23]. In addition, Boeremia was another abundant genus, with 15 strains belonging to Boeremia. Species of Boeremia are morphologically similar to Phoma, and belonged to the Phoma genus previously. Species of Boeremia have been isolated from plants as pathogens or endophytes with a worldwide occurrence, mainly associated with rots of various organs [86]. One of the most well-known varieties is B. exigua var. exigua, which is reported to be a pathogen of more than 200 plant species, causing leaf blight, leaf spot, and connection to post-harvest diseases [87]. The genus was also reported to be isolated from sweet potatoes with leaf spot symptoms in China and Brazil [88,89]. In this study, two new varieties were described, namely B. exigua var. vulgaris and B. exigua var. kasensis.
Didymellaceae is an important group of fungi with a wide host distribution, and legumes, grasses, Asteraceae, buttercups, Rosaceae, and Solanaceae are the most common hosts [22,24,25,27,90]. Chen et al. [25] carried out a correlation analysis between Didymellaceae and host plants. The results indicated that only a few genera showed some degree of host specificity, e.g., Ascochyta exclusively infected legumes, and Gramineae and Cruciferae were host-specific, with Neoascochyta and Phomatodes as hosts, respectively. In the current study, the strains of Didymellaeae had no obvious host specificity. Fifty-one strains were isolated from 12 host genera of nine families, and plants of Haloragidaceae, Hippuridaceae, Hydrocharitaceae, and Potamogetonaceae were common hosts (Table S2). At the species level, Hippuris vulgaris was the most common host, with 25 isolates belonging to 14 species of four genera inhabiting this plant, showing that endophytes from aquatic plants had no host specificity in our study. In terms of the frequency of fungi isolated from the three plant tissues (roots, stems, and leaves), fungi were isolated more frequently from stems and leaves than from roots (Table S2), possibly because roots inhabit an anoxic environment compared to the living environment of stems and leaves.
In our isolates, we found three new species, S. bungeiana, L. shangrilensis, and L. kasensis, characterized by their teleomorphic stage. The Leptosphaerulina species usually have small, dark pseudothecia with membraneous walls and hyaline, and dictyosporous ascospores [59]. Excluding L. saccharicola and L. australis, Leptosphaerulina species are often observed in the teleomorphic stages [74,91]. Stagonosporopsis is mainly regarded as a causal agent in different plants, such as gummy stem blight in pumpkins and ray blight in pyrethrum [92,93]. Typical characteristics of Stagonosporopsis species are their oblong to ellipsoid conidia, as well as the sub-globose conidiomata with slight papillate [82]. In addition, S. malaiana was identified and introduced as a new species belonging to this genus.
In the last decade, our knowledge of the Didymellaceae fungi and their relationships with plant hosts has grown exponentially due to advances in bioinformatics and molecular phylogenetics. As a family of phytopathological importance, they have an important impact on agricultural production. Therefore, it is important to reveal a large number of species and to enrich the species resources in this family. In the present study, a large-scale survey of the Didymellaceae fungi was carried out, demonstrating the presence of a large number of unknown species in some previously neglected ecosystems, and illustrating that the diversity of this family in aquatic plants is much higher than currently estimated. Despite the success of combining four loci (LSU, ITS, rpb2 and tub2), to analyze the phylogenetic relationships of Didymellaceae, there are still some classification statuses to be further determined, such as complex species in Didymella and Boeremia exigua varieties. More genetic loci and isolates are needed to elucidate in detail their phylogenetic relationships and their species boundaries.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof9070761/s1, Table S1: GenBank accession numbers of taxa used in phylogenetic analyses; Table S2: Detailed information on samples of 51 isolates in Didymellaceae.  Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: All sequence data are available in NCBI GenBank following the accession numbers listed in the manuscript.