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 (Hyde et al., 2020b), 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. (2017, 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).

Leptosphaeriaceae (Pleosporales, Dothideomycetes) was established by Barr (1987) and typified by Leptosphaeria. Leptosphaeriaceae is characterized by immersed, erumpent to superficial ascomata, scleroplectenchymatous peridium, cylindrical asci and hyaline to brown, transversely septate ascospores with coelomycetous or hyphomycetous asexual morphs (Alves et al., 2013; de Gruyter et al., 2013; Hyde et al., 2013; Ariyawansa et al., 2015). Historic reviews of Leptosphaeriaceae were detailedly provided by Hyde et al. (2013) and Ariyawansa et al. (2015). Species of Leptosphaeriaceae are widely distributed on various hosts and different regions (Dayarathne et al., 2015; Tennakoon et al., 2017; Phookamsak et al., 2019). They can be saprobic, hemibiotropic, pathogenic, or parasitic occurring on stems and leaves of herbaceous or woody plants in terrestrial and aquatic habitats (Alves et al., 2013; Hyde et al., 2013; Jones et al., 2015; Wanasinghe et al., 2016; Doilom et al., 2018). The early classification of taxa in Leptosphaeriaceae lacked DNA sequence data from ex-type strains. In addition, most strains in GenBank are named without a link to voucher specimens, which is not practical to verify their morphological characteristics to ensure accurate naming (Ariyawansa et al., 2015). Thus, phylogenetic analyses of taxa in Leptosphaeriaceae formed a paraphyletic clade (Dong et al., 1998; Zhang et al., 2012). Ariyawansa et al. (2015) provided a well-resolved backbone tree for Leptosphaeriaceae to resolve species and genera based on multilocus phylogeny with detailed morphology, and the results supported the monophyletic nature of 10 genera in Leptosphaeriaceae among the other families in Pleosporales. Currently, 14 genera are accepted in the family, viz., Alloleptosphaeria, Alternariaster, Chaetoplea, Heterosporicola, Leptosphaeria, Neoleptosphaeria, Ochraceocephala, Paraleptosphaeria, Plenodomus, Pseudoleptosphaeria, Querciphoma, Sclerenchymomyces, Sphaerellopsis, and Subplenodomus (Hongsanan et al., 2020a; Wijayawardene et al., 2020).

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.

Materials and Methods

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.

Fungal Preservation and Fungal Registration Numbers

The herbaria were deposited at the herbarium of Cryptogams, Kunming Institute of Botany Academia Sinica (HKAS), Kunming, Yunnan Province, China and Key Laboratory of Industrial Microbiology and Fermentation Technology of Yunnan (YMF), Kunming, Yunnan Province, China. Living cultures were deposited at the Kunming Institute of Botany Culture Collection (KMUCC), Kunming, Yunnan Province, China. Facesoffungi (FoF) numbers and Index Fungorum (IF) numbers were registered as described by Jayasiri et al. (2015) and Index Fungorum (2021), respectively.

DNA Extraction, PCR Amplification, and Sequencing

Fungi were grown on PDA for 1 week at room temperature (25–30°C). Fungal mycelia were then scraped off and transferred to 1.5 ml sterilized micro-centrifuge tubes. Biospin Fungus Genomic DNA Extraction Kit–BSC14S1 (BioFlux®, China) was used to extract genomic DNA following the manufacturer’s protocol. The LSU 28S rRNA, the SSU 18S rRNA, the ITS, partial translation elongation factor 1-alpha (tef1-α) and partial RNA polymerase II second largest subunit (rpb2) were amplified and sequenced using primers LR0R/LR5 (Vilgalys and Hester, 1990; Rehner and Samuels, 1994), NS1/NS4, ITS5/ITS4 (White et al., 1990), EF1-983F/EF1-2218R (Rehner and Buckley, 2005), and fRPB2-5f/fRPB2-7cR (Liu et al., 1999), respectively.

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 ddH2O. 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).

TABLE 1

GenBank accession numbers and culture collection numbers of species included in the present phylogenetic study.

Species	Culture collection/voucher no.	GenBank accession numbers
		LSU	SSU	ITS
Alloleptosphaeria italica	MFLUCC 14-0934T	KT454714	N/A	KT454722
Alternariaster bidentis	CBS 134021T	KC609341	N/A	KC609333
Alternariaster centaureae-diffusae	MFLUCC 14-0992T	KT454715	KT454730	KT454723
	MFLUCC 15-0009	KT454716	KT454731	KT454724
Alternariaster helianthi	CBS 327.69	KC584369	KC584627	KC609335
Didymella exigua	CBS 183.55NT	EU754155	EU754056	GU237794
Heterosporicola chenopodii	CBS 115.96	EU754188	EU754089	JF740227
	CBS 448.68ET	EU754187	EU754088	FJ427023
Heterosporicola dimorphospora	CBS 165.78	JF740281	JF740098	JF740204
	CBS 345.78ET	GU238069	GU238213	NR111618
Leptosphaeria cichorii	MFLUCC 14-1063T	KT454712	KT454728	KT454720
Leptosphaeria conoidea	CBS 616.75	MH872726	JF740099	MH860957
Leptosphaeria doliolum	MFLU 15-1875	KT454719	KT454734	KT454727
	CBS 155.94	JF740282	N/A	JF740207
	CBS 505.75IT	GQ387576	GQ387515	JF740205
	CBS 541.66	JF740284	N/A	JF740206
Leptosphaeria slovacica	CBS 125975	JF740316	N/A	JF740248
	CBS 389.80	JF740315	JF740101	JF740247
Neoleptosphaeria rubefaciens	CBS 223.77	JF740312	N/A	JF740243
	CBS 387.80	JF740311	N/A	JF740242
Ochraceocephala foeniculi	CBS 145654T	MN516774	MN516743	MN516753
Paraleptosphaeria dryadis	CBS 643.86	GU301828	KC584632	JF740213
Paraleptosphaeria macrospora	CBS 114198	MH874520	N/A	MH862957
Paraleptosphaeria nitschkei	CBS 306.51ET	JF740308	N/A	JF740239
	MFLU 13-0688	KR025864	N/A	KR025860
Paraleptosphaeria orobanches	CBS 101638T	JF740299	N/A	JF740230
Paraleptosphaeria praetermissa	CBS 114591	JF740310	N/A	JF740241
Paraleptosphaeria rubi	MFLUCC 14-0211T	KT454718	KT454733	KT454726
Paraphoma radicina	CBS 111.79ET	EU754191	EU754092	FJ427058
Phaeosphaeria oryzae	CBS 110110ET	GQ387591	GQ387530	KF251186
Plenodomus agnitus	CBS 121.89	JF740271	N/A	JF740194
	CBS 126584	JF740272	N/A	JF740195
Plenodomus artemisiae	KUMCC 18-0151T	MK387958	MK387928	MK387920
	KUMCC 20-0200A	MT957055	MT957048	MT957062
	KUMCC 20-0200B	MT957056	MT957049	MT957063
Plenodomus biglobosus	CBS 119951	JF740274	JF740102	JF740198
	CBS 127249	JF740275	N/A	JF740199
Plenodomus chrysanthemi	CBS 539.63T	GU238151	GU238230	NR111622
Plenodomus collinsoniae	CBS 120227	JF740276	N/A	JF740200
Plenodomus confertus	CBS 375.64	JF740277	N/A	AF439459
Plenodomus congestus	CBS 244.64T	JF740278	N/A	AF439460
Plenodomus deqinensis	CGMCC 3.18221T	KY064031	N/A	KY064027
Plenodomus enteroleucus	CBS 142.84ET	JF740287	N/A	JF740214
	CBS 831.84	JF740288	N/A	JF740215
Plenodomus fallaciosus	CBS 414.62	JF740292	N/A	JF740222
Plenodomus guttulatus	MFLU 15-1876T*	KT454713	KT454729	KT454721
Plenodomus hendersoniae	CBS 113702	MH874506	N/A	MH862939
Plenodomus influorescens	CBS 143.84	JF740297	N/A	JF740228
Plenodomus libanotidis	CBS 113795	MH874508	N/A	MH862943
Plenodomus lijiangensis	KUMCC 18-0186T	MK387959	MK387929	MK387921
Plenodomus lindquistii	CBS 386.80	JF740301	N/A	JF740232
Plenodomus lingam	CBS 260.94	JF740307	N/A	JF740235
Plenodomus lupini	CBS 248.92	JF740303	N/A	JF740236
Plenodomus pimpinellae	CBS 101637T	MH874352	N/A	JF740240
Plenodomus salviae	MFLUCC 13-0219T	KT454717	KT454732	KT454725
Plenodomus sinensis	KUMCC 18-0152	MK387961	MK387931	MK387923
	KUMCC 18-0153	MK387960	MK387930	MK387922
	KUMCC 20-0204	MT957057	MT957050	MT957064
	KUN-HKAS 102227	MK387962	MK387932	MK387924
	MFLU 17-0757PT*	MF072718	MF072720	MF072722
	MFLU 17-0767T*	MF072717	MF072719	MF072721
Plenodomus tracheiphilus	CBS 127250	JF740318	N/A	JF740250
Plenodomus triseptatus	MFLUCC 17-1345T	MN648451	MN648453	MN648452
Plenodomus visci	CBS 122783T	EU754195	EU754096	NR119957
Plenodomus wasabiae	CBS 120119	JF740323	N/A	JF740257
Praeclarispora artemisiae	KUMCC 20-0201AT	MT957053	MT957046	MT957060
	KUMCC 20-0201BT	MT957054	MT957047	MT957061
Pseudoleptosphaeria etheridgei	CBS 125980T	JF740291	N/A	JF740221
Querciphoma carteri	CBS 101633	GQ387593	GQ387532	KF251210
	CBS 105.91	GQ387594	GQ387533	KF251209
Sclerenchymomyces clematidis	MFLUCC 17–2180T	MT214558	MT226675	MT310605
Sclerenchymomyces jonesii	MFLUCC 16-1442T	KY211870	KY211871	KY211869
Sphaerellopsis artemisiae	KUMCC 20-0202AT	MT957058	MT957051	MT957065
	KUMCC 20-0202BT	MT957059	MT957052	MT957066
Sphaerellopsis filum	CBS 234.51	KP170723	N/A	KP170655
	CBS 235.51	KP170724	N/A	KP170656
	CBS 317.68NT	KP170725	N/A	KP170657
Sphaerellopsis hakeae	CPC 29566T	KY173555	N/A	KY173466
Sphaerellopsis isthmospora	HKAS 102225AT*	MK387963	MK387933	MK387925
	HKAS 102225BT*	MK387964	MK387934	MK387926
Sphaerellopsis macroconidialis	CBS 233.51	KP170726	N/A	KP170658
	CBS 658.78T	KP170727	N/A	KP170659
	CPC 21113	KP170728	N/A	KP170660
Sphaerellopsis paraphysata	CPC 21841T	KP170729	N/A	KP170662
	KUMCC 18-0195	MK387965	MK387935	MK387927
Subplenodomus apiicola	CBS 285.72	GU238040	GU238211	JF740196
Subplenodomus drobnjacensis	CBS 270.92	JF740286	N/A	JF740212
Subplenodomus valerianae	CBS 499.91	JF740319	N/A	JF740252
	CBS 630.68	GU238150	GU238229	JF740251
Subplenodomus violicola	CBS 306.68	GU238156	GU238231	FJ427083

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 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).

Results

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.

Phylogenetic analysis of combined LSU, SSU, and ITS sequence data (Figure 1) showed that Praeclarispora artemisiae (KUMCC 20-0201A and KUMCC 20-0201B) clustered with Ochraceocephala (MP/ML/BI = 61%/96%/1.00) in the family Leptosphaeriaceae. Two strains, KUMCC 20-0200A and KUMCC 20-0200B, grouped with the ex-type strain of Plenodomus artemisiae (KUMCC 18-0151) with high bootstrap support (MP/ML/BI = 96%/95%/1.00). The collection KUMCC 20-0204 clustered with MFLU 17-0757 (paratype) and other strains of Plenodomus sinensis (MP/ML/BI = 70%/85%/1.00). Sphaerellopsis artemisiae (KUMCC 20-0202A and KUMCC 20-0202B) grouped separately from its closest relative Sphaerellopsis isthmospora with strong bootstrap support (MP/ML/BI = 100%/100%/1.00) (Figure 1).

FIGURE 1

Phylogenetic tree generated from maximum likelihood analysis (RAxML) based on a combined LSU, SSU, and ITS sequence data. The tree is rooted to Didymella exigua (CBS 183.55). Maximum parsimony and maximum likelihood bootstrap values ≥60% and Bayesian posterior probabilities ≥0.95 (MPBS/MLBS/BYPP) are indicated at the nodes. Ex-epitype, ex-isotype, ex-neotype, ex-type, holotype, and paratype are bolded black, and the new isolates are in red.

Taxonomy

Doilom, W. Dong, K. D. Hyde & C. F. Liao, gen. nov.

Index Fungorum number: IF558142; FoF number: FoF 09225

Etymology: The generic epithet “Praeclarispora” refers to remarkable-spored.

Saprobic on dead twigs of Artemisia argyi. Sexual morph: Ascomata black, scattered to gregarious, breaking the epidermis in linear fissures, semi-immersed, becoming erumpent to superficial, subglobose, uni- to multi-loculate, coriaceous, with ostiolate papilla. Peridium unevenly relatively thick, composed of several layers of thick-walled cells of textura angularis, outer layer black, inner layer brown. Hamathecium comprising numerous, filiform, septate, of cellular pseudoparaphyses embedded in a gelatinous matrix. Asci eight-spored, bitunicate, fissitunicate, narrowly obovoid, short pedicellate, apically rounded, with ocular chamber. Ascospores tri- to tetra-seriate, fusiform, curved, tapered toward the acute ends, versicolor, 0–1-septate when immature, becoming brown in median cell and hyaline to pale brown in other cells, middle cell larger than other cells, septate when mature, slightly constricted at the septa, thin- and smooth-walled. Asexual morph: Undetermined.

Type species: P. artemisiae Doilom, W. Dong, K. D. Hyde and C. F. Liao

Notes: Based on a blastn search of NCBIs GenBank, the closest hits using LSU sequence of Praeclarispora artemisiae matches with several genera in Leptosphaeriaceae and has highest similarity to Sphaerellopsis filum (CBS 234.51, identities = 99.18%) and S. paraphysata (CPC 21841, identities = 99.05%), followed by Ochraceocephala foeniculi (CBS 145654, identities = 98.83%). The closest hits using SSU sequence are Plenodomus lingam (CBS 260.94, identities = 99.81%), Pl. artemisiae (KUMCC 20-0200A, identities = 99.81%), Pl. biglobosus (CBS 119951, identities = 99.81%), and O. foeniculi (CBS 145654, identities = 99.71%). ITS sequence matches with published species Pl. hendersoniae (CBS 113702, identities = 92.14%), Pl. biglobosus (CBS 119951, identities = 89.66%), and O. foeniculi (CBS 145654, identities = 87.93%). However, in our multilocus analysis (Figure 1), P. artemisiae forms a sister branch with O. foeniculi with 96% ML and 1.00 BYPP but low MP bootstrap support, and separates from Leptosphaeria, Plenodomus, and Sphaerellopsis. The ITS phylogeny has similar results with the multilocus phylogeny (Supplementary Figure 1); LSU phylogenetic analysis clearly shows Praeclarispora separates from O. foeniculi as a distinct genus (Supplementary Figure 2). A single gene comparison between P. artemisiae and O. foeniculi shows that there are 1.17% (10/854), 0.29% (3/1029), and 13.81% (76/550) nucleotide difference in LSU, SSU, and ITS sequence data, respectively.

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.

Doilom, W. Dong, K. D. Hyde & C. F. Liao, sp. nov., Figure 2.

FIGURE 2

Praeclarispora artemisiae (HKAS 112654, holotype). (a–c) Appearance of ascomata on host substrate. (d,e) Vertical sections through ascomata. (f) Pseudoparaphyses. (g–i) Asci. (j–n) Ascospores. (o) Germinated ascospore. (p) Colony on PDA after 15 days (above and below views). Scale bars: (c) = 250 μm, (d) = 50 μm, (e) = 30 μm, (f,k–m) = 10 μm, (g–j,n,o) = 20 μm.

Index Fungorum number: IF558143; FoF number: FoF 09226

Etymology: The specific epithet “artemisiae” refers to the host genus Artemisia.

Holotype: HKAS 112654

Saprobic on dead twigs of Artemisia argyi. Sexual morph: Ascomata 170–245 μm high, 185–285 μm diam., black, scattered to gregarious, breaking the epidermis in linear fissures, semi-immersed, becoming erumpent to superficial, subglobose, uni- to multi-loculate, coriaceous, with ostiolate papilla. Ostioles 50–70 μm diam., central, brown, ostiolar canal filled with periphyses. Peridium 30–60 μm wide at the sides, unevenly thick, composed of scleroplectenchymatous cells, arrange in textura angularis, outer layer black, inner layer brown. Hamathecium 2–4.5 μm diam., numerous, filiform, septate, of cellular pseudoparaphyses embedded in a gelatinous matrix. Asci 100–140 × 19–27 μm ( = 120 × 23 μm, n = 15), eight-spored, bitunicate, fissitunicate, narrowly obovoid, short pedicellate, apically rounded, with ocular chamber. Ascospores 55–70 × 6–11 μm ( = 61 × 8.5 μm, n = 20), overlapping tri- to tetra-seriate, fusiform, curved, tapered to acute ends, versicolor, 0–1-septate when immature, becoming brown in median cell or occasionally two median cells, and hyaline to pale brown in other cells (Figure 2g), middle cell longer and slightly wider than the other cells, 8–10(–12)-euseptate when mature, slightly constricted at the septa, guttulate, thin- and smooth-walled. Asexual morph: Undetermined.

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.

Material examined: CHINA, Yunnan Province, Qujing City, dead twigs of Artemisia argyi (Asteraceae), October 1, 2019, C. F. Liao, (HKAS 112654, holotype); ex-type living culture KUMCC 20-0201; ibid., YMF 107390, isotype.

Additional GenBank numbers: tef1-α = MW396658 (KUMCC 20-0201A); MW396659 (KUMCC 20-0201B).

A. Karunarathna, Phookamsak and K. D. Hyde, in Phookamsak et al., Fungal Diversity 95: 23 (2019), Figures 3, 4a

FIGURE 3

Plenodomus artemisiae (HKAS 112653, new collection). (a,b) Ascomata on host substrate. (c,d) Vertical sections of ascomata. (e) Structure of peridium. (f) Pseudoparaphyses. (g–i) Asci. (j–l) Ascospores (l showing the old ascospore occasionally with 5–7 septa). (m) Germinated ascospore. Scale bars: (c,d) = 50 μm, (e) = 30 μm, (f–m) = 10 μm.

FIGURE 4

Colonies on PDA (up-front, down-reverse). (a) Plenodomus artemisiae (HKAS 112653). (b) Plenodomus sinensis (HKAS 112657). (c) Sphaerellopsis artemisiae (HKAS 112655).

Saprobic on dead stems of Artemisia argyi. Sexual morph: Ascomata 140–230 μm high, 210–260 μm diam., black, scattered, superficial with base seated in the substrate, compressed globose, uniloculate, glabrous, coriaceous, ostiolate, obscurely papillate. Peridium unevenly thick, 20–60 μm wide at the sides, with a poorly developed base, 10–15 μm wide, thinner toward the papilla, 12–15 μm wide, composed of several layers of thick-walled cells of textura angularis, outer layer black, inner layer brown. Hamathecium 2–2.5 μm diam., numerous, filiform, hyaline, septate, of cellular pseudoparaphyses embedded in a gelatinous matrix. Asci 60–80 × 9.5–11 μm ( = 68 × 10 μm, n = 15), eight-spored, bitunicate, fissitunicate, cylindric-clavate, short pedicellate, apically rounded. Ascospores 29–36 × 4.5–5.5 μm ( = 33 × 4.8 μm, n = 15), overlapping 2–3-seriate, narrowly fusiform, with tapering and rounded ends, hyaline, 1-septate, occasionally 5–7-septate when old, constricted at the median septum, guttulate, thin- and smooth-walled, without sheath or appendages. Asexual morph: Undetermined.

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.

Material examined: CHINA, Yunnan Province, Qujing City, dead stems of Artemisia argyi (Asteraceae), October 1, 2019, C. F. Liao, (HKAS 112653, new collection); living culture KUMCC 20-0200; ibid., YMF 112653, new collection.

Additional GenBank numbers: tef1-α = MW396660 (KUMCC 20-0200A); MW396661 (KUMCC 20-0200B).

Notes: Our collections KUMCC 20-0200A and KUMCC 20-0200B cluster with Plenodomus artemisiae (KUMCC 18-0151) with strong bootstrap support (MP/ML/BI = 99%/96%/1.00) (Figure 1). They have very similar morphological characteristics, except our collections have more septa (5–7-septate vs. only 5-septate) than the holotype (KUN-HKAS 102226) of Pl. artemisiae (Phookamsak et al., 2019). Our two collections have 99.65%, 99.90, 99.44, and 99.34% similarities with the ex-type strain (KUMCC 18-0151) of Pl. artemisiae in LSU, SSU, ITS, and tef1-α sequence data, respectively, which indicate them to be conspecific. We therefore identify our collections as Pl. artemisiae based on morphological and molecular evidences. Plenodomus artemisiae was initially reported from dead branches and stems of an unidentified Artemisia sp. in Yunnan Province, China (Phookamsak et al., 2019). Our new collection was collected from the same region, and we identify its host as Artemisia argyi.

Tennakoon, Phook. and K. D. Hyde, in Tennakoon et al., Phytotaxa 324(1): 76 (2017), Figures 4b, 5

FIGURE 5

Plenodomus sinensis (HKAS 112657, new host record). (a) Ascomata on host substrate. (b–d) Vertical section of ascomata. (e) Structure of peridium. (g–i) Asci. (f,j–l) Ascospores (l Ascospore in Indian Ink). (m) Germinated ascospore. Scale bars: (b–d) = 50 μm, (e) = 30 μm, (f,m) = 20 μm, (g–l) = 10 μm.

Saprobic on Ageratina adenophora. Sexual morph: Ascomata 220–325 μm high, 205–345 μm diam., black, scattered to gregarious, raised, superficial, subglobose to conical, with flattened and thickened base, uniloculate, glabrous, coriaceous, with minutely ostiolate papilla, easily removed from the host substrate. Ostioles 55–95 μm diam., central, brown, ostiolar canal filled with some periphyses. Peridium unevenly thick, 25–115 μm wide at the base, 30–70 μm wide at the sides, mostly thickened at the base and thinner at the sides, three-layered, outer layer composed of dark brown, thick-walled cells of textura angularis, middle layer composed of pale brown to subhyaline, thick-walled, large cells of textura globulosa or textura angularis, inner layer composed of light brown, thin-walled, compressed cells of textura angularis. Hamathecium 2–4.5 μm wide, septate, branched, of cellular pseudoparaphyses embedded in a gelatinous matrix, slightly constricted at the septa. Asci 80–115 × 10–13 μm ( = 98 × 11 μm, n = 20), eight-spored, bitunicate, fissitunicate, cylindrical, with short furcate pedicel, apically rounded, with a distinct ocular chamber. Ascospores 29–39 × 4–5.5 μm ( = 33 × 4.9 μm, n = 30), overlapping 2–3-seriate, hyaline, 0–1-septate when immature, becoming olivaceous to yellowish, fusiform, with obtuse ends, 6–7-septate, constricted at the middle septum, not or slightly constricted at each septum, cell above central septum slightly wider, guttulate, thick- and smooth-walled, with mucilaginous globoid-shaped appendages at both ends. Asexual morph: Undetermined.

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.

Material examined: CHINA, Yunnan Province, Chuxiong City, Daguokou Township, Biji Village, dead branches of Ageratina adenophora (Asteraceae), September 14, 2019, C. F. Liao, (HKAS 112657, new host record); living culture KUMCC 20-0204; ibid., YMF 107633, new host record.

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.

Doilom, W. Dong, K. D. Hyde and C. F. Liao, sp. nov., Figures 4c, 6

FIGURE 6

Sphaerellopsis artemisiae (HKAS 112655, holotype). (a, b) Ascomata on host substrate. (c,d) Vertical sections of ascomata. (e) Structure of peridium. (f) Splitted ascospores. (g–i) Asci. (j–l) Ascospores. (m) Germinated ascospore. Scale bars: (c,d) = 50 μm, (e) = 30 μm, (f–m) = 20 μm.

Index Fungorum number: IF557892; FoF number: FoF 09227

Etymology: The specific epithet “artemisiae” refers to the host genus Artemisia.

Holotype: HKAS 112655

Saprobic on dead stems of Artemisia argyi. Sexual morph: Ascomata 320–400 μm high, 230–300 μm diam., black, scattered or gregarious in small groups, superficial, subglobose, uniloculate, glabrous, coriaceous, with minutely ostiolate papilla. Ostioles 110–130 μm diam., central, dark brown to black, ostiolar canal filled with some periphyses. Peridium 30–55 μm at the sides, unevenly thick, thicker at the base, up to 70 μm wide, thinner at the ostiole, 13–18 μm wide, composed of several layers of brown to dark brown, thin-walled, large cells of textura angularis, inwardly compressed. Hamathecium 2–5.5 μm wide, sparse, hyaline, filamentous, septate, cellular pseudoparaphyses, constricted at the septa. Asci 105–170 × 17–25.5 μm ( = 122 × 20.5 μm, n = 15), eight-spored, bitunicate, fissitunicate, narrowly clavate, short pedicellate, apically rounded with a well-developed ocular chamber. Ascospores 80–117 × 5–7.5 μm ( = 92.5 × 6.5 μm, n = 25), overlapping 4–6-seriate, hyaline to yellowish, scolecosporous, bent at the fourth to fifth septum from the base, 10–13-septate, constricted at the septa, split into two part-spores at the bending point when old; upper part 45–75 μm long, cylindrical, 6–8-septate, with rounded apex and truncate base; lower part 30–42 μm long, subcylindric-clavate, 3–4-septate, with truncate apex and tapering or conical base, guttulate, without sheath or appendages. Asexual morph: Undetermined.

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.

Material examined: CHINA, Yunnan Province, Kunming City, dead stems of Artemisia argyi (Asteraceae), October 27, 2019, C. F. Liao, (HKAS 112655, holotype), ex-type living culture KUMCC 20-0202; ibid., YMF 107391, isotype.

Additional GenBank numbers: tef1-α = MW396662 (KUMCC 20-0202A); = MW396663 (KUMCC 20-0202B).

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 (Hyde et al., 2019). 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 thick-walled 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 (Lumbsch and 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 (Hyde et al., 2020a). Our collection amends the morphology of P. sinensis, which is useful for fungal identification.

Additional protein-coding markers such as rpb2 and tef1-α 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.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.