The Plant Family Asteraceae Is a Cache for Novel Fungal Diversity: Novel Species and Genera With Remarkable Ascospores in Leptosphaeriaceae

In a cursory survey of fungi on Asteraceae in Yunnan Province, China, we report fungal species belonging to the family Leptosphaeriaceae (Pleosporales, Dothideomycetes). Two novel species have remarkable ascospores that are unusual for sexual ascomycetes. Multilocus phylogeny of large subunit, small subunit, and internal transcribed spacer sequence data showed one to be a novel genus, while the other is a new species. Praeclarispora artemisiae gen. et sp. nov. is introduced and is typical of Leptosphaeriaceae, but has unusual fusiform, versicolor ascospores with a brown median cell. Sphaerellopsis artemisiae sp. nov. has scolecosporous ascospores with deeply constricted septa that split into two parts, which resembles S. isthmospora but differs by ascospore dimension and molecular data. In addition, Plenodomus artemisiae is reported as a new collection from dead stems of Artemisia argyi in Qujing City. Plenodomus sinensis is reported as a new host record from Ageratina adenophora. All taxa are illustrated and described based on evidence of taxonomy and phylogeny.


INTRODUCTION
The plant family Asteraceae (= Compositae) is the major and widespread family of Angiosperms (flowering plants). The family comprises over 1,900 genera with over 32,000 accepted species (The Plant List, 2013). Most members of Asteraceae are herbaceous plants, but a significant number are shrubs, climbers, and trees. The family has a cosmopolitan distribution ranging from subpolar to tropical regions. The largest proportion of species occurs in arid and semiarid regions of subtropical and lower to middle temperate latitudes (Barkley et al., 2006). Several members of Asteraceae are economically important plants as food crops, including globe artichoke (Cynara cardunculus var. scolymus), lettuce (Lactuca spp.), safflower (Carthamus tinctorius), and sunflower (Helianthus spp.). Many genera are important in horticulture such as pot marigold (Calendula officinalis) and coneflowers (Echinacea spp.), and others are of herbal medicinal importance, including gumweed (Grindelia spp.), yarrow (Achillea millefolium), and silvery wormwood (Artemisia argyi). Many species in Asteraceae are also considered as invasive weeds including sticky snakeroot (Ageratina adenophora) and Siam weed (Chromolaena odorata).
Studying the fungi on Asteraceae will provide important information toward establishing the numbers of fungi , due to the fact that many species are herbaceous in arid and semi-arid areas, where we know little about fungal diversity. Several novel fungal species have been described from the plant family Asteraceae. For example, Hermatomyces chromolaenae from stems of Chromolaena odorata, Torula chromolaenae from a dead branch of C. odorata, and Dendryphion hydei from branch litter of Bidens pilosa were introduced by Li et al. (2017Li et al. ( , 2020 and Tibpromma et al. (2017), respectively. A novel genus, Neocochlearomyces isolated from leaves of C. odorata, was described by Crous et al. (2018). Phookamsak et al. (2019) introduced novel fungal species from Cirsium arvense and Artemisia sp. Mapook et al. (2020) introduced 60 new taxa from Siam weed, including one new family Neomassarinaceae, 12 new genera, and 47 new species. Herein, fungal species belonging to the family Leptosphaeriaceae are reported from Ageratina adenophora and Artemisia argyi (Asteraceae).
In this study, we introduce a new genus and two new species from Artemisia argyi that have remarkable ascospores. In addition, Plenodomus sinensis is reported as a new host record from Ageratina adenophora. Combined analyses of large subunit (LSU), small subunit (SSU), and internal transcribed spacer (ITS) sequence data with morphology supported the placement of our taxa in Leptosphaeriaceae.

Sample Collection, Specimen Examination, and Fungal Isolation
The specimens of Ageratina adenophora and Artemisia argyi belonging in Asteraceae were collected from Yunnan Province, China. Specimens were placed in zip-lock plastic bags and returned to the laboratory for fungal observation and isolation. Fungal structures on the host substrates were observed using the Motic SMZ 161 stereomicroscope and their ascomata on substrates were captured with a digital camera fitted on to the stereomicroscope. Micro-morphological characteristics were observed and photographed with a Nikon ECLIPSE Ni compound microscope fitted with a Canon EOS 600D digital camera. Indian Ink was used to observe mucilaginous sheaths surrounding the ascospores. Micro-morphological characteristics were measured by the Tarosoft (R) Image Frame Work program. Images used for figures were edited with Adobe Photoshop CS6 software (Adobe Systems, United States).
Fungal isolation was made from single spore as detailed in Chomnunti et al. (2014). Germinating ascospores were observed using the Motic SMZ 161 stereomicroscope and single ascospore was transferred using sterile needle and grown on potato dextrose agar (PDA) at room temperature (25-30 • C). Pure cultures were kept for further studies.
The PCR amplification was performed in a total volume of 25 µl. PCR mixtures contained 12.5 µl of Easy Taq PCR Super Mix, 1 µl of dNTPs, 1 µl of each primer, and 9.5 µl of ddH 2 O. The PCR thermal cycle program for LSU, SSU, and ITS amplification was provided as initially 94 • C for 3 min, followed by 35 cycles of denaturation at 94 • C for 30 s, annealing at 56 • C for 50 s, elongation at 72 • C for 90 s, and a final extension at 72 • C for 10 min. The annealing was adjusted to 52 • C and 55 • C for rpb2 and tef1-α, respectively. PCR products were purified and sequenced at Shanghai Sangon Biological Engineering Technology & Services Co., (Shanghai, China). GenBank accession numbers of tef1-α of our strains are provided in "Additional GenBank numbers."

Phylogenetic Analysis
Consensus sequences were generated using BioEdit v.7.2.5 (Hall, 1999). Sequences of each strain were blasted using the MegaBLAST search of GenBank's nucleotide database 1 to examine their closest taxa. A total 89 sequences were used in phylogenetic analyses (Table 1). Didymella exigua (CBS 183.55) was used as the outgroup taxon. Individual dataset of the LSU, SSU, and ITS was aligned online with MAFFT version v.7.471 (Katoh et al., 2019) 2 and manually edited where necessary using BioEdit v.7.2.5. Phylogenetic trees were inferred with maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI).
Maximum parsimony analysis was performed with PAUP v. 4.0b10, with the parameter setting as the method described in Wanasinghe et al. (2018). Descriptive tree statistics for parsimony [Tree Length (TL), Consistency Index (CI), Retention Index (RI), Relative Consistency Index (RC), and Homoplasy Index (HI)] were calculated for trees generated under different optimality criteria. ML analysis was calculated as the method described in Doilom et al. (2017). All free model parameters will be estimated by RAxML and ML estimate of 25 per site rate categories. The model selected for ML was GTRGAMMA. BI analysis was conducted using the Markov Chain Monte Carlo (MCMC) method with MrBayes v. 3.2.7 (Huelsenbeck and Ronquist, 2001). By using MrModeltest 2.2 (Nylander, 2004), the GTR + I + G was selected as the best-fit nucleotide 1 http://blast.ncbi.nlm.nih.gov/ 2 http://mafft.cbrc.jp/alignment/server/index.html substitution models under the Akaike information criterion (AIC) for LSU, SSU, and ITS sequence data. Six chains were run for the individual and combined datasets. The MCMC algorithm was started from a random tree topology. Five million generations were selected with a sampling frequency every 100 generations. The Tracer v.1.6 program (Rambaut et al., 2013) was used to check the effective sampling sizes (ESS) that should be above 200, the stable likelihood plateaus, and burn-in value. The results suggest that the first 5,000 generations should be excluded as burn-in. Phylogenetic trees were visualized using FigTree v.1.4.0 (Rambaut, 2009) and formatted using PowerPoint 2010 (Microsoft Corporation, WA, United States).

Phylogenetic Analysis
The alignment comprised 90 strains including the outgroup taxon, which consisted of 3,286 characters including alignment gaps (1-1331 bp for LSU, 1332-2680 bp for SSU, and 2681-3286 bp for ITS). The MP analysis for the combined dataset had 325 parsimony informative, 2,840 constant, and 121 parsimony uninformative characters and yielded 18 most parsimonious trees (TL = 2158, CI = 0.342, RI = 0.729, HI = 0.658, and RC = 0.249). The RAxML analysis resulted in a best scoring likelihood tree selected with a final combined dataset = −15205.152646. The matrix had 620 distinct alignment patterns, with 36.47% of undetermined characters or gaps.
Ochraceocephala foeniculi is only known from its hyphomycetous asexual morph, which is characterized by hyaline, loosely or densely branched conidiophores, phialidic conidiogenous cells, and hyaline to yellowish, globose to subglobose conidia, and isolated as plant pathogen from living Foeniculum vulgare (Aiello et al., 2020). Praeclarispora artemisiae is reported herein from only its ascomycetous sexual morph, characterized by black ascomata, narrowly obovoid asci, and fusiform ascospores with a larger, brown, median cell, and isolated as saprobe from decaying twigs of Artemisia argyi. Unfortunately, we could not obtain the asexual morph from the culture for further morphological assessments. Even though we observed them under different conditions as described in Phookamsak et al. (2015) and Senanayake et al. (2020), neither conidia nor conidiomatal structures were produced. Therefore, we believe that it is wise to keep Ochraceocephala and Praeclarispora as separate genera in Leptosphaeriaceae for now. A different scenario may occur with the discovery of similar fungi from both of their asexual and sexual morphs with more fresh sampling.
Culture characteristics: On PDA, colony circular, reaching 45 mm diam. in 15 days at room temperature (25-30 • C), surface rough, with sparse mycelia on the surface, dry, umbonate from the side view, edge entire; from above, yellowish to cream at the margin, gray at the middle, white at the center; from below, yellowish at the margin, gray at the middle, yellowish brown at the center; producing yellowish pigmentation in culture.
Culture characteristics: On PDA, colony irregular, reaching 40 mm diam. in 14 days at room temperature (25-30 • C), surface rough, with dense mycelia, velvety and fluffy, dry, raised from the side view, edge undulate; from above, yellowish at the margin, cream to white at the center; from below, yellowish at the margin, orange brown at the middle, black at the center; producing yellowish pigmentation in culture.
Culture characteristics: On PDA, colony irregular, reaching 40 mm diam. in 30 days at room temperature (25-30 • C), surface rough and dull, with dense mycelia mostly immersed in culture, dry, umbonate from the side view, edge undulate; from above, dark gray at the margin, gray at the center; from below, greenish at the margin, black at the center; not producing pigmentation in culture.
Notes: Our specimen HKAS 112657 and the holotype of Plenodomus sinensis (MFLU 17-0767) have 6-7-septate ascospores with mucilaginous globoid-shaped appendages at both ends, but they are slightly different in ascomatal base. Tennakoon et al. (2017) described flattened ascomatal base in the holotype, in addition, we observed the thickened one in our collection. Multilocus phylogeny shows that our collection KUMCC 20-0204 clusters with four collections of Pl. sinensis, including paratype MFLU 17-0757, but separates from the holotype MFLU 17-0767. Although MFLU 17-0767 clusters with Pl. collinsoniae (Figure 1), it differs in having larger asci, longer ascospores with olivaceous to yellowish pigmentation as discussed in Tennakoon et al. (2017). Our collection must be Pl. sinensis as its morphological characteristics are more similar to Pl. sinensis. Plenodomus sinensis appears to have a wide host range, occurring on Cirsium sp., Plukenetia volubilis, Tamarindus indica, and ferns in China (Tennakoon et al., 2017;Phookamsak et al., 2019). This is the first report of Pl. sinensis on Ageratina adenophora in China.
Culture characteristics: On PDA, colony circular, reaching 15 mm diam. in 7 days at room temperature (25-30 • C), surface rough, with dense mycelia, velvety to fluffy, dry, raised from the side view, edge entire; from above, white to cream; from below, white at the margin, pale brown at the middle, black at the center; not producing pigmentation in culture.
Notes: In our multilocus analysis, our collections Sphaerellopsis artemisiae (KUMCC 20-0202A and KUMCC 20-0202B) cluster with Sphaerellopsis isthmospora and separate from other Sphaerellopsis species with high bootstrap support (Figure 1). Sphaerellopsis artemisiae resembles S. isthmospora in having scolecosporous ascospores with deeply constricted septa that split into two parts at the fourth to fifth septum from the base (Phookamsak et al., 2019), but it differs in having longer and wider ascospores (92.5 × 6.5 µm vs. 87.1 × 5.9 µm). Phylogenetic analysis of combined LSU, SSU, and ITS sequence data also supports the idea that they are different species (Figure 1). In addition, a comparison of tef1-α sequence data shows that S. artemisiae has 4.04% differences with S. isthmospora. Based on morphological difference and molecular data, we therefore introduce S. artemisiae as a novel species.

DISCUSSION
The members of the plant family Asteraceae are distributed throughout the world. Many novel fungal species have been reported from several genera in this family (Li et al., 2017;Tibpromma et al., 2017;Crous et al., 2018;Phookamsak et al., 2019;Mapook et al., 2020). Thus, Asteraceae is a promising cache of novel fungal species that warrant further study for basic science, use in biocontrol and biotechnology . Our study reveals one new genus (Praeclarispora), two new species (Praeclarispora artemisiae and Sphaerellopsis artemisiae), one new collection of the sexual morph report (Plenodomus artemisiae), and one new host record (Pl. sinensis) on Ageratina adenophora in Yunnan Province, China. The two new species have remarkable ascospores that are unusual for sexual ascomycetes when compared with other genera (Doilom et al., 2018;Pem et al., 2019;Dong et al., 2020;Hongsanan et al., 2020a,b;Hyde et al., 2020c).
Praeclarispora has fusiform ascospores, with a larger median cell and tapering end cells which is slightly similar to Heptameria. However, Praeclarispora and Heptameria are different genera based on the distinct characteristics of ascomata, asci and ascospores. Heptameria has pseudothecial ascomata with rather thick pseudothecial wall (100-160 µm thick in H. obesa) (Lucas and Sutton, 1971), whereas Praeclarispora has euthecial ascomata with relatively thin peridium (30-60 µm thick in P. artemisiae). In addition, Heptameria often forms in several roundish groups on the substrate (Lucas and Sutton, 1971), while Praeclarispora mostly forms in linear fissures (never form in roundish groups). Heptameria has club-like asci (Lucas and Sutton, 1971), while they are narrowly obovoid in Praeclarispora. The ascospores of Heptameria are bi-or tri-seriate in the upper portion of the asci and uniseriate below (Lucas and Sutton, 1971), contrasting the tri-to tetra-seriate ascospores in Praeclarispora. Heptameria has distoseptate, rather thickwalled ascospores with a median, brown, rather large and muriform cell comprising of several transverse, longitudinal, and occasionally oblique septa (Lucas and Sutton, 1971), while Praeclarispora has euseptate, thin-walled ascospores and lacking the muriform median cell. Unfortunately, Heptameria cannot be incorporated in the phylogenetic tree as lacking sequence data and is referred to Dothideomycetes genera incertae sedis based on morphology Huhndorf, 2007, 2010;Zhang et al., 2012;Hyde et al., 2013;Wijayawardene et al., 2020). On the other hand, the available sequence data support Praeclarispora as a distinct genus within Leptosphaeriaceae (Figure 1).
Heptameria was introduced by Thümen with H. elegans as the type species (Thümen, 1879). However, H. elegans was considered as a synonym of an earlier proposed species H. obesa (≡ Sphaeria obesa) based on the examination of the holotype of H. elegans and H. obesa (Lucas and Sutton, 1971). Therefore, H. obesa is used as the type species. Although the current name of H. obesa is recorded as Leptosphaeria obesa in Index Fungorum (2021), Heptameria is not synonymized as Leptosphaeria and treated as a distinct genus by its cucurbitaria-like pseudothecia and characteristic ascospores (Petrak, 1951), which is also accepted by recent outline of fungi (Wijayawardene et al., 2020). However, sequence data derived from the type species H. obesa are indeed needed to confirm whether Heptameria is a valid genus, as most species of Heptameria have been transferred to other genera. Currently, only two species, i.e., H. obesa and H. uncinata, are accepted in the genus (Lucas and Sutton, 1971). It is very likely that the type species H. obesa will be extinct as it has been missing for nearly 150 years, especially in the increasingly serve climate change. Considering this circumstance and avoiding future confusion of Heptameria, Praeclarispora gen. nov. is introduced based on its distinct morphology.
One of the findings here is that Plenodomus sinensis has both flattened and thickened ascomatal bases, while the type that was studied by Tennakoon et al. (2017) has a flattened ascomatal base. The information of new collections and new records can be used to update fungal classification and improved identification of species . Our collection amends the morphology of P. sinensis, which is useful for fungal identification.
Additional protein-coding markers such as rpb2 and tef1α are necessary to improve the phylogenetic resolution of genera and families in Pleosporales (Jaklitsch et al., 2018). However, most species of Leptosphaeriaceae lack tef1-α sequence data and other protein-coding markers, and some known species were sequenced using different tef1-α primer pairs. Thus, the phylogenetic analysis was constructed based on combined LSU, SSU, and ITS sequence data as provided in Dayarathne et al. (2015); Wanasinghe et al. (2016), Tennakoon et al. (2017), and this study. Nevertheless, we provide tef1-α sequence data for P. artemisiae, P. artemisiae, and S. artemisiae to facilitate the future identification of species. The rpb2 sequence data were unsuccessfully obtained even after several attempts.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

AUTHOR CONTRIBUTIONS
MD and WD designed the study. MD, WD, and KH wrote the manuscript. MD, WD, C-FL, and NS conducted the experiments, analyzed the data, and revised the manuscript. MD, NS, and SL contributed to research funds. All authors revised the manuscript.