Morphological and Phylogenetic Characterisations Reveal Four New Species in Leptosphaeriaceae (Pleosporales, Dothideomycetes)

Abstract

Leptosphaeriaceae is a widely distributed fungal family with diverse lifestyles. The family includes several genera that can be distinguished by morphology and molecular phylogenetic analysis. During our investigation of saprobic fungi on grasslands in Yunnan Province, China, four fungal taxa belonging to Leptosphaeriaceae associated with grasses were collected. Morphological observations and phylogenetic analyses of the combined SSU, LSU, ITS, tub2, and rpb2 loci based on maximum likelihood and Bayesian inference were used to reveal the taxonomic placement of these fungal taxa. This study introduces four new taxa, viz. Leptosphaeria yunnanensis, L. zhaotongensis, Paraleptosphaeria kunmingensis, and Plenodomus zhaotongensis. Colour photo plates, full descriptions, and a phylogenetic tree to show the placement of the new taxa are provided.

1. Introduction

Leptosphaeriaceae, introduced by Barr [1], are widely distributed and exhibit diverse lifestyles, including fungicolous, epiphytic, parasitic, saprobic, and hemibiotrophic on herbaceous and woody plants [2,3,4,5,6]. For example, Leptosphaeria species are saprobes, plant pathogens, or hemibiotrophs on cultivated, wild herbaceous, and woody plants [7,8,9,10,11]. In the order Pleosporales, the family Leptosphaeriaceae contains economically significant plant pathogens [2]: i.e., Zhang et al. [8] reported Leptosphaeria species cause a serious disease of oilseed rape (Brassica napus, canola) in China.

The sexual morphs of Leptosphaeriaceae species are characterised by immersed to superficial and conical or globose ascomata, a thick peridium with scleroplectenchymatous or plectenchymatous cells, cylindrical to oblong asci, and hyaline to brown ascospores that are transversely septate [2,3,6,12,13,14,15]. In addition, asexual morphs are coelomycetous or hyphomycetous [13,16,17]. Furthermore, the most recently evolved Leptosphaeria species produce paler, longer, fusiform, and narrower ascospores with three or multi-septa, compared with the primitive dark brown ascospores [2,5].

Leptosphaeria and Paraleptosphaeria are morphologically similar, but phylogenetic analysis can clearly distinguish them [9,17,18,19,20]. The same applies to Plenodomus, and the other six genera, viz. Alloleptosphaeria, Alternariaster, Neoleptosphaeria, Pseudoleptosphaeria, Sphaerellopsis, and Subplenodomus, that were re-circumscribed based on molecular phylogeny [2]. Based on the multilocus phylogeny of the combined LSU, SSU, and ITS datasets, seven other genera, viz. Angularia, Chaetoplea [21], Ochraceocephala [22], Praeclarispora [23], Heterosporicola, Querciphoma, and Sclerenchymomyces [6], have been described in Leptosphaeriaceae. The Index Fungorum [24] listed 1682 Leptosphaeria species (accessed on 29 March 2023) epithets, and many of them have been synonymised under other genera. The members of Paraleptosphaeria have been reported as saprobic, fungicolous, or pathogenic [2,4,20], and nine species epithets are listed in the Index Fungorum [24] (accessed on 29 March 2023). In addition, Plenodomus has often been reported as saprobic or pathogenic on Asteraceae, Fabaceae, Lamiaceae, Liliaceae, and Poaceae [2,6,13,25,26] and 101 records are listed in the Index Fungorum [24] (accessed on 29 March 2023).

Grasslands comprise a biome subjected to alternating drought, where grass and grass-like species dominate, and arboreous trees are uncommon [27]. In the grassland biome, several living organisms, such as herbivorous mammals, insects, and fungi (pathogenic, saprobic, and symbiotic), play essential roles in maintaining biomass and biodiversity [28]. Regarding fungi, a checklist of Ascomycetes on grasses was provided by Karunarathna et al. [29], which lists 3,165 fungal species. Studies of fungi on grasses include those of Thambugala et al. [30], Goonasekara et al. [31], and Brahmanage et al. [32].

This study describes four new Leptosphaeriaceae species that were collected from herbaceous plants in Kunming and Zhaotong, Yunnan Province, China. Phylogenetic analyses results based on SSU, LSU, ITS, tub2, and rpb2 loci, colour photo plates, complete descriptions of the four new species, and a summary of the morphological characteristics of Leptosphaeria, Paraleptosphaeria, and Plenodomus are provided.

2. Materials and Methods

2.1. Sample Collection, Isolation, and Identification

Herbaceous plants with fungal fruiting bodies were collected from Kunming and Zhaotong in Yunnan Province, China, stored in plastic bags, and returned to the mycology laboratory at the Kunming Institute of Botany. The samples were examined under an Olympus SZ-61 dissecting microscope (Tokyo, Japan). Fungal fruiting bodies were manually sectioned and mounted in double distilled water (ddH₂O). Micro-morphological characteristics were captured using an OLYMPUS SZ2-ILST compound microscope connected to an Industrial Digital Camera 16NP USB3.0 (Panasonic, Osaka, Japan) microscope imaging system. Photo plates were processed using Adobe Photoshop CS6 Extended version 13.0.1 (Adobe Systems, San Jose, CA, USA). As described by Senanayake et al. [33], cultures were obtained via single-spore isolation and incubated under normal light at room temperature (25 °C). Germinating ascospores or conidia were observed under a stereo microscope and transferred to new potato dextrose agar (PDA) plates. Herbarium specimens were deposited in the herbarium at the Kunming Institute of Botany, Chinese Academy of Sciences (HKAS), and the Herbarium Mycologicum Academiae Sinicae, Beijing, China (HMAS), and living cultures were deposited in the China General Microbiological Culture Collection Center (CGMCC). The Index Fungorum [24] and Faces of Fungi (FoF) [34] numbers were registered for the new species.

2.2. DNA Extraction, PCR Amplification, and DNA Sequencing

Genomic DNA was extracted from fresh mycelia grown on PDA at 28 °C for two weeks using the Biospin Fungus Genomic DNA Extraction Ki-BSC14S1 (BioFlux^®, Hangzhou, China) according to the manufacturer’s protocol. The E.Z.N.A. Forensic DNA Kit-D3591 (Omega Biotek, Inc., Norcross, Georgia) was used to extract DNA directly from fruiting bodies. Polymerase chain reactions (PCRs) were carried out for five genetic markers: internal transcribed spacer region (ITS) [35], partial 28S large subunit nuclear ribosomal DNA (LSU) [36], partial small subunit ribosomal RNA (SSU) [35], β-tubulin (tub2) [37], and partial RNA polymerase second largest subunit (rpb2) [38]. The primers and amplification conditions used are listed in

Table 1. Details of genes/loci with PCR primers and thermal cycling program for PCR amplification.

Genes/Loci	PCR Primers (Forward/Reverse)	PCR Thermal Cycle Protocols	References
ITS	ITS5/ITS4	a Annealing at 55 °C for 15 s c	[35]
LSU	LR0R/LR5		[36]
SSU	NS1/NS4	a Annealing at 55 °C for 30 s c	[35]
tub2	T1/Bt2b		[37]
rpb2	fRPB2-5F/fRPB2-7cR	b Annealing at 57 °C for 50 s c	[38]

Notes: ^a initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 10s, elongation at 72 °C for 20 s; ^b initial denaturation at 95 °C for 3 min, followed by 35 cycles at 95 °C for 45 s, elongation at 72 °C for 1.5 min; ^c final extension at 72 °C for 10 min.

. The total volume of PCR mixtures for amplification was 25 μL, containing 8.5 μL ddH₂O, 12.5 μL 2xF8FastLong PCR MasterMix (Beijing Aidlab Biotechnologies Co. Ltd., Beijing, China), 2 μL of the DNA template, and 1 μL each of reverse and forward primer (10 pM). The PCR products were sequenced by Shanghai Sangon Biological Engineering Technology and Service Co., Ltd., Shanghai, China.

2.3. Phylogenetic Analyses

The newly obtained sequences (SSU, LSU, ITS, tub2, and rpb2) were subjected to BLASTn searches against the GenBank database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (accessed on 29 March 2023) to identify closely related taxa. Reference isolates and accessions were obtained from recent studies [6,17,23,39,40,41] and downloaded from GenBank. Single-locus sequence datasets were aligned using MAFFT v. 7.505 [42], trimmed using TrimAl v. 1.3 [43] via the web server Phylemon2 (http://phylemon.bioinfo.cipf.es/utilities.html) (accessed on 29 March 2023), and concatenated using BioEdit v. 7.0.5.2 [44]. Phylogenetic reconstructions of individual and combined datasets were performed using maximum likelihood (ML) and Bayesian inference (BI) analyses.

Maximum likelihood trees were inferred using RAxML-HPC2 in the XSEDE v. 8.2.12 [45] in CIPRES Science Gateway v. 3.3 online platform [46] under the GTRGAMMA nucleotide substitution model with 1000 bootstrap replicates. Bayesian inference analysis was conducted using MrBayes on XSEDE v. 3.2.7a [47], under the substitution model GTR + I + G for all loci, estimated by MrModeltest v. 2.3 [48] using PAUP v. 4.0b10 [49]. Six simultaneous Markov chains were run for 10,000,000 generations, with trees sampled every 1000th generation. The run was configured to stop when the standard deviation of split frequencies dropped below 0.01, and the first 25% of the trees were discarded as burn-in.

Tree topologies were visualised and exported using FigTree v. 1.4.0 [50]. The phylogram was edited and annotated using Microsoft Office PowerPoint 2016 (Microsoft Inc., Redmond, WA, USA) and Adobe Photoshop CS6 Extended version 13.0.1 (Adobe Systems, San Jose, CA, USA). Finally, the newly generated sequences were deposited in the GenBank database (

Table 2. Names, Index Fungorum strain numbers, and corresponding GenBank accession numbers of the taxa used for phylogenetic analyses in this study.

Species Name	Strain Numbers	GenBank Accession Numbers
		ITS	LSU	SSU	tub2	rpb2
Alloleptosphaeria clematidis	MFLUCC 17-2071	MT310604	MT214557	MT226674	NA	NA
All. iridicola	CBS 143395	MH107919	MH107965	NA	NA	NA
All. italica	MFLUCC 14-0934	KT454722	KT454714	NA	NA	NA
All. shangrilana	HKAS 112210	MW431059	MW431315	MW431058	NA	NA
Alternariaster bidentis	CBS 134021	KC609333	KC609341	NA	NA	KC609347
Alt. bidentis	CBS 134185	KC609334	KC609342	NA	NA	KC609348
Alt. centaureae-diffusae	MFLUCC 14-0992	KT454723	KT454715	KT454730	NA	NA
Alt. centaureae-diffusae	MFLUCC 15-0009	KT454724	KT454716	KT454731	NA	NA
Alt. centaureae-diffusae	HMJAU 60188	OL996125	OL897175	OL891810	OL898721	NA
Alt. helianthi	CBS 199.86	KC609336	KC609343	NA	NA	KC609349
Alt. helianthi	CBS 327.69	KC609335	KC584369	KC584627	NA	KC584494
Alt. trigonosporus	MFLU 15-2237	KY674857	KY674858	NA	NA	NA
Angularia xanthoceratis	HMJAU 60197	OM295683	OM295682	OM295681	OM304358	NA
Didymella exigua	CBS 183.55	EF192139	EU754155	EU754056	GU237525	EU874850
D. maydis	CBS 588.69	FJ427086	MH871149	EU754093	FJ427190	GU371782
Heterosporicola beijingensis	JZB3400001	MN733734	MN737597	MN733738	NA	NA
H. beijingensis	JZB3400002	MN733735	MN737598	MN733739	NA	NA
H. beijingensis	JZB3400003	MN733736	MN737599	MN733740	NA	NA
H. beijingensis	JZB3400004	MN733737	MN737600	MN733741	NA	NA
H. chenopodii	CBS 448.68	FJ427023	EU754187	EU754088	NA	NA
H. chenopodii	CBS 115.96	JF740227	EU754188	EU754089	NA	GU371775
H. dimorphospora	CBS 345.78	JF740203	GU238069	GU238213	NA	NA
H. dimorphospora	CBS 165.78	JF740204	JF740281	JF740098	NA	NA
Leptosphaeria chatkalica	YGS22	MW886101	MW886099	MW886100	NA	NA
L. cichorii	MFLUCC 14-1063	KT454720	KT454712	KT454728	NA	NA
L. cirsii	MFLUCC 14-1170	NR155328	NG059725	NA	NA	NA
L. conoidea	CBS 125977	JF740202	JF740280	NA	NA	NA
L. conoidea	CBS 616.75	JF740201	JF740279	JF740099	KT389804	KT389639
L. conoidea	FeF93	MZ492958	NA	NA	NA	NA
L. doliolum	MFLUCC 15-1875	KT454727	KT454719	KT454734	NA	NA
L. doliolum	CBS 155.94	JF740207	JF740282	NA	JF740146	NA
L. doliolum	CBS 505.75	JF740205	GQ387576	GQ387515	JF740144	KY064035
L. doliolum	CBS 541.66	JF740206	JF740284	NA	JF740145	NA
L. doliolum	CBS 130000	JF740210	NA	NA	JF740149	NA
L. ebuli	MFLUCC 14-0828	KP744446	KP744488	KP753954	NA	NA
L. errabunda	CBS 617.75	JF740216	JF740289	NA	JF740150	NA
L. errabunda	CBS 125978	JF740217	JF740290	NA	JF740151	NA
L. errabunda	CBS 129998	JF740219	MH877027	NA	JF740153	NA
L. italica	MFLU15-0174	NA	KT783670	NA	NA	NA
L. irregularis	MFLUCC 15-1118	KX856056	KX856055	NA	NA	NA
L. macrocapsa	CBS 640.93	JF740237	JF740304	NA	JF740156	NA
L. pedicularis	CBS 390.80	JF740224	JF740294	NA	JF740155	NA
L. pedicularis	CBS 126582	JF740223	JF740293	NA	NA	NA
L. proteicola	CPC:18289	JQ044439	JQ044458	NA	NA	NA
L. regiae	MFLUCC 18-1137	MN244201	MN244171	MN244177	NA	NA
L. sclerotioides	CBS 144.84	JF740192	JF740269	NA	NA	NA
L. sclerotioides	CBS 148.84	JF740193	JF740270	NA	NA	NA
L. sclerotioides	FeF422	MZ492959	NA	NA	NA	NA
L. sclerotioides	P10	MT996500	MT996501	NA	MT996502	MT996503
L. sclerotioides	P9	MT996499	MT992704	NA	MT989358	MT992705
Leptosphaeria sp.	LW119	MH128282	NA	NA	NA	NA
Leptosphaeria sp.	LW113	MH128276	NA	NA	NA	NA
L. slovacica	CBS 389.80	JF740247	JF740315	JF740101	NA	NA
L. slovacica	CBS 125975	JF740248	JF740316	NA	NA	NA
L. sydowii	CBS 385.80	JF740244	JF740313	NA	JF740157	NA
L. sydowii	CBS 125976	JF740245	JF740314	NA	JF740158	NA
L. urticae	MFLU 18-0591	MK123333	MK123332	MK123329	NA	NA
L. urticae	FeF166	MZ492960	NA	NA	NA	NA
L. veronicae	CBS 126583	JF740255	JF740321	NA	JF740161	NA
L. veronicae	CBS 145.84	JF740254	JF740320	NA	JF740160	NA
L. yunnanensis	CGMCC 3.23748	OP494319	OP494327	OP494333	OP476696	NA
L. yunnanensis	CGMCC 3.23749	OP494320	OP494328	OP494334	OP476697	NA
L. yunnanensis	HKAS 124671	OP494321	OP494329	OP494335	OP476698	NA
L. zhaotongensis	HKAS 124664	OP494318	OP494326	OP494332	OP476695	NA
L. zhaotongensis	HMAS 352282	OQ446062	OQ446132	OQ448836	OQ511597
Neoleptosphaeria jonesii	MFLUCC 16-1442	KY211869	KY211870	KY211871	NA	NA
N. rubefaciens	CBS 223.77	JF740243	JF740312	NA	NA	NA
N. rubefaciens	CBS 387.80	JF740242	JF740311	NA	NA	NA
Ochraceocephala foeniculi	CBS 145654	MN516753	MN516774	MN516743	MN520147	MN520145
O. foeniculi	Di3AF15	MN516766	MN516783	MN516752	NA	NA
Paraleptosphaeria dryadis	CBS 643.86	JF740213	GU301828	KC584632	NA	GU371733
Pa. dryadis	CBS 743.86	AF439461	NA	NA	NA	NA
Pa. kunmingensis	KUNCC 23-12732	OP494316	OP494324	OP494330	OP476693	OP476691
Pa. kunmingensis	KUNCC 23-12731	OQ446060	OQ446130	OQ448834	OQ511598	OQ455053
Pa. macrospora	CBS 114198	JF740238	JF740305	NA	NA	NA
Pa. nitschkei	CBS 306.51	JF740239	JF740308	NA	KT389833	KT389660
Pa. nitschkei	MFLUCC 13-0688	KR025860	KR025864	NA	NA	NA
Pa. orobanches	CBS 101638	JF740230	JF740299	A	NA	NA
Pa. padi	MFLU 15-2756	KY554203	KY554198	KY554201	NA	NA
Pa. polylepidis	APA-2999	MK795714	MK795717	NA	NA	NA
Pa. praetermissa	CBS 114591	JF740241	JF740310	NA	NA	NA
Pa. rubi	MFLUCC 14-0211	KT454726	KT454718	KT454733	NA	NA
Pa. rumicis	CBS 522.78	KF251144	KF251648	NA	NA	NA
Plenodomus agnitus	CBS 121.89	JF740194	JF740271	NA	KY064053	KY064036
Pl. agnitus	CBS 126584	JF740195	JF740272	NA	NA	NA
Pl. agnitus	MFLU 15-0039	KP744459	KP744504	NA	NA	NA
Pl. artemisiae	KUMCC 18-0151	MK387920	MK387958	MK387928	NA	MK435607
Pl. artemisiae	KUMCC 20-0200A	MT957062	MT957055	MT957048	NA	NA
Pl. artemisiae	KUMCC 20-0200B	MT957063	MT957056	MT957049	NA	NA
Pl. biglobosus	CBS 119951	JF740198	JF740274	JF740102	KY064054	KY064037
Pl. biglobosus	CBS 127249	JF740199	JF740275	NA	NA	NA
Pl. changchunensis	CCMJ5011	OL996123	OL897174	OL984031	NA	NA
Pl. changchunensis	CCMJ5012	OL996124	OL966928	OL984032	OL898716	OL944508
Pl. collinsoniae	VT02	MN653010	MN982862	MN652269	NA	NA
Pl. collinsoniae	CBS 120227	JF740200	JF740276	NA	KY064056	KY064039
Pl. collinsoniae	KNU-AP100C	LC550566	LC550568	NA	NA	NA
Pl. collinsoniae	KNU-20-A1	LC591836	NA	NA	LC591846	LC591841
Pl. collinsoniae	KNU-20-A2	LC591837	NA	NA	LC591847	LC591842
Pl. collinsoniae	KNU-20-A3	LC591838	NA	NA	LC591848	LC591843
Pl. collinsoniae	KNU-20-A4	LC591839	NA	NA	LC591849	LC591844
Pl. collinsoniae	KNU-20-C4	LC591840	NA	NA	LC591850	LC591845
Pl. collinsoniae	FeC109	MW446975	NA	NA	NA	NA
Pl. collinsoniae	EF194	MK842112	NA	NA	NA	NA
Pl. confertus	CBS 375.64	AF439459	JF740277	NA	KY064057	KY064040
Pl. congestus	CBS 244.64	AF439460	JF740278	NA	KY064058	KY064041
Pl. deqinensis	CGMCC 3.18221	KY064027	KY064031	NA	KY064052	KY064034
Pl. dezfulensis	IRAN 4159C	MZ048609	NA	NA	MZ043102	MZ043104
Pl. dezfulensis	SCUA-Ahm-S41-2	MZ048610	NA	NA	MZ043103	MZ043105
Pl. enteroleucus	CBS 142.84	JF740214	JF740287	NA	KT266266	KY064042
Pl. enteroleucus	CBS 831.84	JF740215	JF740288	NA	KT266270	NA
Pl. enteroleucus	F-146,176	MN910295	MN910294	NA	NA	NA
Pl. enteroleucus	ICMP:10937	KT309810	KT309635	NA	KT309399	NA
Pl. fallaciosus	CBS 414.62	JF740222	JF740292	NA	NA	KY064043
Pl. guttulatus	MFLUCC 15-1876	KT454721	KT454713	KT454729	NA	NA
Pl. hendersoniae	CBS 113702	JF740225	JF740295	NA	KT266271	KY064044
Pl. hendersoniae	CBS 139.78	JF740226	JF740296	NA	NA	NA
Pl. hendersoniae	LTO	MF795790	NA	NA	NA	MF795832
Pl. influorescens	CBS 143.84	JF740228	JF740297	NA	KT266267	KY064045
Pl. influorescens	PD 73/1382	JF740229	JF740298	NA	KT266273	NA
Pl. libanotidis	CBS 113795	JF740231	JF740300	NA	KY064059	KY064046
Pl. lijiangensis	KUMCC 18-0186	MK387921	MK387959	MK387929	NA	NA
Pl. lindquistii	CBS 386.80	JF740232	JF740301	NA	NA	NA
Pl. lindquistii	CBS 381.67	JF740233	JF740302	NA	NA	NA
Pl. lindquistii	MF-Ha 16-005	MK495988	NA	NA	MK501790	NA
Pl. lingam	AFTOL-ID 277	KT225526	DQ470946	DQ470993	NA	DQ470894
Pl. lingam	CBS 275.63	JF740234	JF740306	JF740103	KT389841	KT389669
Pl. lingam	CBS 260.94	JF740235	JF740307	NA	MZ073915	KY064047
Pl. lingam	CBS 147.24	MH854784	MH866288	NA	MZ073914	NA
Pl. lupini	CBS 248.92	JF740236	JF740303	NA	KY064061	KY064048
Pl. pimpinellae	CBS 101637	JF740240	JF740309	NA	KY064062	NA
Pl. salviae	MFLUCC 13-0219	KT454725	KT454717	KT454732	NA	NA
Pl. sinensis	KUMCC 18-0152	MK387923	MK387961	MK387931	NA	NA
Pl. sinensis	KUMCC 18-0153	MK387922	MK387960	MK387930	NA	MK435608
Pl. sinensis	KUN-HKAS 102227	MK387924	MK387962	MK387932	NA	NA
Pl. sinensis	MFLUCC 17-0757	MF072722	MF072718	MF072720	NA	NA
Pl. sinensis	MFLUCC 17-0767	MF072721	MF072717	MF072719	NA	NA
Pl. sinensis	KNU-GW1901	LC550567	LC550569	LC550570	NA	NA
Pl. sinensis	KUMCC 20-0204	MT957064	MT957057	MT957050	NA	NA
Pl. tracheiphilus	CBS 551.93	JF740249	JF740317	JF740104	MZ073918	KY064049
Pl. tracheiphilus	CBS 127250	JF740250	JF740318	NA	MZ073919	NA
Pl. tracheiphilus	MUCL 38481	MW810293	MW715037	NA	MZ073920	NA
Pl. tracheiphilus	ATCC 26007	MZ049614	MW959165	NA	MZ073908	MZ073893
Pl. tracheiphilus	IS3-15	MK461058	NA	NA	NA	NA
Pl. triseptatus	MFLUCC 17-1345	MN648452	MN648451	MN648453	NA	NA
Pl. visci	CBS 122783	JF740256	EU754195	EU754096	KY064063	KY064050
Pl. visci	CPC:35314	MT223830	MT223922	NA	NA	MT223696
Pl. visci	CPC:35315	MT223831	MT223923	NA	NA	NA
Pl. visci	CPC:35316	MT223832	MT223924	NA	NA	NA
Pl. wasabiae	CBS 120119	JF740257	JF740323	NA	KT266272	NA
Pl. wasabiae	CBS 120120	JF740258	JF740324	NA	NA	NA
Pl. zhaotongensis	CGMCC 3.23746	OP494317	OP494325	OP494331	OP476694	OP476692
Pl. zhaotongensis	CGMCC 3.23747	OQ446061	OQ446131	OQ448835	OQ511599	OQ455054
Praeclarispora artemisiae	KUMCC 20-0201A	MT957060	MT957053	MT957046	NA	NA
Pr. artemisiae	KUMCC 20-0201B	MT957061	MT957054	MT957047	NA	NA
Pseudoleptosphaeria etheridgei	CBS 125980	JF740221	JF740291	NA	NA	MT394686
Querciphoma carteri	CBS 101633	KF251210	GQ387593	GQ387532	KF252701	NA
Querciphoma carteri	CBS 105.91	KF251209	GQ387594	GQ387533	KF252700	NA
Sclerenchymomyces clematidis	MFLUCC 17-2180	MT310605	MT214558	MT226675	NA	NA
Sphaerellopsis artemisiae	KUMCC 20-0202A	MT957065	MT957058	MT957051	NA	NA
Sp. artemisiae	KUMCC 20-0202B	MT957066	MT957059	MT957052	NA	NA
Sp. filum	CBS 234.51	KP170655	KP170723	NA	KP170704	NA
Sp. filum	CBS 235.51	KP170656	KP170724	NA	KP170705	NA
Sp. filum	CBS 317.68	KP170657	KP170725	NA	KP170706	NA
Sp. hakeae	CPC:29566	KY173466	KY173555	NA	NA	NA
Sp. isthmospora	HKAS 102225A	MK387925	MK387963	NA	NA	NA
Sp. isthmospora	HKAS 102225B	MK387926	MK387964	MK387934	NA	NA
Sp. macroconidialis	CBS 233.51	KP170658	KP170726	NA	KP170707	NA
Sp. macroconidialis	CBS 658.78	KP170659	KP170727	NA	KP170708	NA
Sp. macroconidialis	CPC:21113	KP170660	KP170728	NA	KP170709	NA
Sp. paraphysata	CPC:21841	KP170662	KP170729	NA	KP170710	NA
Sp. paraphysata	KUMCC 18-0195	MK387927	MK387965	MK387935	NA	NA
Subplenodomus apiicola	CBS 285.72	JF740196	GU238040	GU238211	NA	NA
Su. apiicola	CBS 421.50	MH856699	MH868215	NA	NA	NA
Su. drobnjacensis	CBS 270.92	JF740212	JF740286	NA	NA	NA
Su. drobnjacensis	CBS 269.92	JF740211	JF740285	JF740100	NA	NA
Su. galicola	MFLU 15-1368	KY554204	KY554199	NA	NA	NA
Su. valerianae	CBS 630.68	JF740251	GU238150	GU238229	NA	NA
Su. valerianae	CBS 499.91	JF740252	JF740319	NA	NA	NA
Su. violicola	CBS 306.68	FJ427083	GU238156	GU238231	KT389849	NA
Shiraia bambusicola	GZAAS2.0703	GQ845412	KC460981	NA	NA	NA
Shiraia bambusicola	GZAAS2.0629	GQ845415	KC460980	NA	NA	NA
Tzeanania taiwanensis	NTUCC 17-005	MH461123	MH461120	MH461126	MH461132	NA
Tzeanania taiwanensis	NTUCC 17-006	MH461124	MH461121	MH461127	MH461133	NA

Notes: The ex-types are in bold, and newly generated sequences are shown in blue. NA: sequence data are not available.

).

The decision as to whether the new species should be introduced followed the polyphasic guidelines of Chethana et al. [51] and Maharachchikumbura et al. [52].

3. Results

3.1. Phylogenetic Analyses

The combined sequence data of SSU, LSU, ITS, tub2, and rpb2 consisted of 177 strains of Leptosphaeriaceae, plus Didymella exigua (CBS 183.55) and D. maydis (CBS 588.69) as outgroup taxa (Figure 1). After trimming, the dataset consisted of 3981 sites, including gaps (SSU = 1–894 bp, LSU = 895–2224 bp, ITS = 2225–2780 bp, tub2 = 2781–3108 bp, and rpb2 = 3109–3981 bp). The phylogenetic tree topologies of the single and combined matrices were similar. The phylogenetic topologies obtained using the ML and BI methods also shared the same topology. RAxML analysis of the combined dataset yielded a best-scoring tree with a final ML optimization likelihood value of −31,560.945159. The matrix had 1315 distinct alignment patterns, with 45.47% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.243300, C = 0.230842, G = 0.272923, T = 0.252935; substitution rates were as follows: AC = 1.583725, AG = 4.540133, AT = 1.817809, CG = 0.949937, CT = 7.364395, GT = 1.00, alpha: 0.147156.

Our phylogenetic analysis of Leptosphaeriaceae is analogous to the analysis by Xu et al. [6]. Our isolate, Leptosphaeria zhaotongensis (HKAS 124664, HMAS 352282), is closely related to L. cichorii (MFLUCC 14-1063) with 96% ML and 1.00 BYPP bootstrap support (Figure 1). Leptosphaeria yunnanensis (CGMCC 3.23748, CGMCC 3.23749, HKAS 124671) formed a well-separated lineage from other Leptosphaeria species with 67% ML and 0.91 BYPP bootstrap support (Figure 1). Plenodomus zhaotongensis (CGMCC 3.23746, CGMCC 3.23747) is closely related to the Pl. agnitus strains with 91% ML and 1.00 BYPP bootstrap support (Figure 1). Paraleptosphaeria kunmingensis (KUNCC 23-12732, KUNCC 23-12731) is closely related to Pa. macrospora (CBS 114198) with 88% ML and 1.00 BYPP bootstrap support (Figure 1).

3.2. Taxonomy

3.2.1. Leptosphaeria yunnanensis Y. Gao and H. Gui, sp. nov.

Index Fungorum number: IF 556122; Faces of Fungi number: FoF 12905; Figure 2.

Etymology: The specific epithet “yunnanensis” refers to Yunnan Province, where the holotype was collected.

Holotype: HKAS 124670

Saprobic on a decaying stalk of herbaceous plant. Sexual morph: Undetermined. Asexual morph: Conidiomata 280–515 μm diam × 170–370 μm high ($\stackrel{-}{\mathrm{x}}$ = 385 × 253 μm, n = 20), in small groups or scattered, solitary, erumpent to superficial, subglobose to globose, smooth-walled, easily removed from the host substrate, black, coriaceous, without ostiolate. Conidiomatal wall (132–)144–189(–202) μm thick, ($\stackrel{-}{\mathrm{x}}$ = 166 μm, n = 30), thick, almost fills the entire conidiomata, each cell-layer (9–)10.6–15.6(–18) μm wide, ($\stackrel{-}{\mathrm{x}}$ = 13 μm, n = 40), composed of flattened cells of textura angularis, highly pigmented on the outside and hyaline on the inside, with conidiogenous cells. Conidiogenous cells (2.8–)3.6–5.2(–6.4) μm long × (3.8–)4.4–5.8(–6.7) μm wide ($\stackrel{-}{\mathrm{x}}$ = 4.4 × 5 μm, n = 40), ampulliform or globose to subglobose, smooth-walled, hyaline. Conidia (2.5–)3.3–5.6(–6.6) μm long × (1–)1.2–1.5(–1.8) μm wide ($\stackrel{-}{\mathrm{x}}$ = 4.5 × 1.4 μm, n = 30), ellipsoidal to sub-cylindrical with obtuse ends, hyaline, guttulate, aseptate, smooth-walled.

Culture characteristics: Conidia germinated on PDA within 20 h. Colonies on PDA reaching 20 mm at four weeks at room temperature (25–27 °C), hairy or cottony, raised, white to grey, mycelium superficial, dark brown at the margin, white to light grey at the centre from above, grey in the centre, gradually black towards the edges from below.

Material examined: China, Yunnan Province, Zhaotong City, Daguan County, grassland (27°44′23″ N, 103°47′59″ E), on a decaying stalk of herbaceous plant, 21 August 2021, Ying Gao, ZG25A (HKAS 124670, holotype), ex-type, CGMCC 3.23748; ibid., ZG25C (HKAS 127125, paratype), ex-paratype CGMCC 3.23749; ibid., ZG25 (HKAS 124671, paratype).

Notes: Based on multilocus phylogenetic analyses, our isolates of L. yunnanensis (CGMCC 3.23748, CGMCC 3.23749, and HKAS 124671) showed a well-separated lineage within Leptosphaeria with moderate statistical support (67% ML, 0.91PP (Figure 1)). It clustered between L. urticae (MFLU 18-0591) and L. pedicularis (CBS 390.80) (Figure 1). The pairwise nucleotide comparison showed that our strain (CGMCC 3.23748) differs from L. urticae (MFLU 18-0591) in 44/487 bp of ITS (9.03%, with 5 gaps) and L. pedicularis (CBS 390.80) in 37/513 bp of ITS (7.23%, with 6 gaps) and 24/334 bp of tub2 (7.19%, with 2 gaps). Significant morphological differences were observed when compared with the literature for the genus (for example, [2,5,13,41,53]). Leptosphaeria yunnanensis differs from related species by having a unique conidiomatal wall that occupies almost the entire interior of the conidiomata and sporulation indistinctly in the centre. In addition, Leptosphaeria yunnanensis was reported as an asexual form, and although it is similar to L. cichorii in conidiomata and conidiogenous cells, the sizes are different (conidiomata 280–515 × 170–370 μm vs. 189–200 × 196–220 μm) (conidiogenous cells 2.8–6.5 × 3.5–6.5 μm vs. 2–5 × 2–4 μm) [2]. Furthermore, these two species formed a well-separated lineage (Figure 1). Therefore, based on the polyphasic approach recommended for species-boundary delimitation [51,52], we introduce L. yunnanensis as a novel taxon.

3.2.2. Leptosphaeria zhaotongensis Y. Gao and H. Gui, sp. nov.

Index Fungorum number: IF 556123; Faces of Fungi number: FoF 12904; Figure 3.

Etymology: The specific epithet “zhaotongensis” refers to Zhaotong City, where the holotype was collected.

Holotype: HKAS 124664

Saprobic on a decaying stalk of a herb. Sexual morph: Ascomata 380–550 μm diam × 185–300 μm high ($\stackrel{-}{\mathrm{x}}$ = 461 × 234 μm, n = 15), scattered or in small groups, solitary, erumpent to superficial, globose to ampulliform, smooth-walled, easily removed from the host substrate, with a flattened base, black, coriaceous, uni-loculate, glabrous, ostiolate. Ostiole apex conical and with papilla. Peridium (35–)66–116.5(–127) μm wide, ($\stackrel{-}{\mathrm{x}}$ = 91 μm, n = 40), thick-walled, composed of 4–8 layers of flattened, light brown to dark brown cells of textura angularis. Hamathecium (1.5–)2–2.6(–3) μm wide, ($\stackrel{-}{\mathrm{x}}$ = 2.3 μm, n = 50), straight, septate, hyaline, branched, cellular pseudoparaphyses, partially embedded in a gelatinous matrix. Asci (84–)92–113(–118) × (7.7–)8.2–10(–11) μm ($\stackrel{-}{\mathrm{x}}$ = 102 × 9 μm, n = 30), eight-spored, arising from the base, cylindrical to cylindric-clavate, bitunicate, fissitunicate, apically rounded, short pedicellate with foot-like pedicel, with ocular chamber, hyaline. Ascospores (16.2–)17.6–20(–21.3) × (4.5–)4.8–5.7(–6.5) μm ($\stackrel{-}{\mathrm{x}}$ = 18.7 × 5.2 μm, n = 40), uniseriate, fusiform, partially overlapping, narrow to acute at both ends, guttulate, initially hyaline with one septum, becoming yellowish to brown, 3-septate at maturity, broader cells above central septum, often slightly constricted at septum, sometimes rough-walled, without mucilaginous sheath. Asexual morph: Undetermined.

Material examined: China, Yunnan Province, Zhaotong City, Daguan County, grassland (27°44′23″ N, 103°47′59″ E), on a decaying stalk of herbaceous plant, 21 August 2021, Ying Gao, GG (HKAS 124664, holotype); ibid., (HMAS 352282, paratype).

Notes: Based on our phylogenetic analysis of the combined SSU, LSU, ITS, tub2, and rpb2 sequence data, our novel species L. zhaotongensis (HKAS 124664, HMAS 352282) is closely related to L. cichorii (MFLUCC 14-1063) with 96% ML and 1.00 PP statistical support (Figure 1). Leptosphaeria zhaotongensis differs from L. cichorii in its larger ascomata (384–551 × 186–292 μm vs. 206–240 × 251–363 μm) and ascospores (16–21 × 5–7 μm, hyaline, yellowish to brown, guttulate vs. 11–20 × 3–6 μm, reddish to yellowish brown, without guttulate). The similarity of the ITS sequence data of L. cichorii (MFLUCC 14-1063) was 62/521 bp (11.9%, with 6 gaps) compared to L. cichorii (MFLUCC 14-1063). Therefore, based on the polyphasic approach recommended for species boundaries delimitation [51,52], we introduce L. zhaotongensis as a novel taxon.

3.2.3. Paraleptosphaeria kunmingensis Y. Gao and H. Gui, sp. nov.

Index Fungorum number: IF 556124; Faces of Fungi number: FoF 12902; Figure 4.

Etymology: The specific epithet “kunmingensis” refers to Kunming City, where the holotype was collected.

Holotype: HKAS 124662

Saprobic on a decaying stalk of herbaceous plant. Sexual morph: Ascomata 215–300 μm × 145–220 μm ($\stackrel{-}{\mathrm{x}}$ = 253 × 176 μm, n = 15), scattered, gregarious, immersed in the epidermis of the host, globose or subglobose and flat-globose, dark brown to black, uni-loculate, glabrous, shiny, papillate, with ostiole. Peridium (21–)24–34(–43) μm wide, ($\stackrel{-}{\mathrm{x}}$ = 29 μm, n = 35), composed of 2–4 layers of flattened, light brown to dark brown cells of textura angularis. Hamathecium (1.6–)2–3(–3.7) μm wide, ($\stackrel{-}{\mathrm{x}}$ = 2.5 μm, n = 40), straight, septate, hyaline, unbranched, cellular pseudoparaphyses, embedded in a gelatinous matrix. Asci (70–)73–92(–104) × (12–)13–15.7(–16.4) μm ($\stackrel{-}{\mathrm{x}}$ = 83 × 14 μm, n = 25), eight-spored, arising from base, fissitunicate, bitunicate, cylindrical to cylindric-clavate, short pedicellate with club-like pedicel, thick-walled at the apex, hyaline, with ocular chamber. Ascospores (33.5–)37.6–47(–50.5) × (5–)5.2–6.2(–7.2) μm ($\stackrel{-}{\mathrm{x}}$ = 42.3 × 5.7 μm, n = 30), overlapping, 2–3-seriate, hyaline, guttulate, lunate to long fusiform or inequilateral, straight or slightly curved, with 1–3 transverse septa, often slightly constricted at septum, swollen at the second cell, rounded to slightly pointed at both ends, guttulate, without a mucilaginous sheath. Asexual morph: Undetermined.

Culture characteristics: Ascospores germinated on PDA within 20 h, and a germ tube was initially produced from the middle. Colonies on PDA reaching 15 mm at two weeks at room temperature, circular, slightly raised, curled, floccose, pale yellow from above, dark brown in the centre, gradually pale yellow towards the edges from below, grows towards the filamentous edge.

Material examined: China, Yunnan Province, Kunming City, (25°8′19″ N, 102°44′25″ E), on a decaying stalk of herbaceous plant, 20 June 2021, Ying Gao, CCSG18A (HKAS 124662, holotype), ex-type KUNCC 23-12732. ibid., CCSG18 (HKAS 127126, paratype), ex-paratype KUNCC 23-12731.

Notes: Paraleptosphaeria kunmingensis is introduced as a new species based on its distinct morphology and phylogenetic analysis of combined SSU, LSU, ITS, tub2, and rpb2 datasets. Paraleptosphaeria kunmingensis is closely related to Pa. macrospora (CBS 114198) with 88% ML and 1.00 BYPP bootstrap support (Figure 1). The species differs from Pa. macrospora (Basionym: Metasphaeria macrospora) in its smaller asci (83 × 14 μm vs. 105 × 18 μm), smaller ascospores (42.3 × 5.7 μm vs. 44 × 8 μm), and the number of septa of ascospores (1–3 septa vs. three septa) [13,54]. In addition, the ITS pairwise nucleotide comparison of these species showed 18/523 bp differences (3.44%, without gaps). Our isolate differs from Pa. nitschkei in 7.51% (KT389833) and Pa. dryadis (GU371733) in 10.05% in the tub2 and rpb2 regions, respectively. Therefore, based on the polyphasic approach recommended for species boundaries delimitation [51,52], we introduce Pa. kunmingensis as a novel taxon.

3.2.4. Plenodomus zhaotongensis Y. Gao and H. Gui, sp. nov.

Index Fungorum number: IF 556124; Faces of Fungi number: FoF 12903; Figure 5.

Etymology: The specific epithet “zhaotongensis” refers to Zhaotong City, where the holotype was collected.

Holotype: HKAS 124668

Saprobic on a decaying stalk of herbaceous plant. Sexual morph: Ascomata 200–300 μm × 210–320 μm ($\stackrel{-}{\mathrm{x}}$ = 240 × 298 μm, n = 15), scattered, most are gregarious, raised, superficial with base seated in the substrate, globose to subglobose or irregular, apically conical, dark brown to black, uni-loculate, glabrous, coriaceous, ostiolate, papillate ostiole, wider and flattened at the base, connected by very thin stromatal tissue at the base. Peridium (22–)29–54(–79) μm wide, ($\stackrel{-}{\mathrm{x}}$ = 42 μm, n = 50), composed of two types of scleroplectenchymatous cells layers, thick-walled of unequal thickness, thickened at base, thinner toward sides and apex, inner layers composed of hyaline to pale brown cells of textura angularis to textura globulosa, outer layer of amorphous black cells. Hamathecium (1.4–)2–3.7(–6) μm wide, ($\stackrel{-}{\mathrm{x}}$ = 3 μm, n = 45), septate, hyaline, unbranched, broad at base, tapering upwards, pseudoparaphyses. Asci (80–)97–120(–132) × (10–)11–13(–14) μm ($\stackrel{-}{\mathrm{x}}$ = 109 × 12 μm, n = 20), eight-spored, arising from base, fissitunicate, bitunicate, cylindric-clavate, initially hyaline, short pedicellate with foot-like pedicel, with ocular chamber, thick-walled at the apex. Ascospores (34–)35–39(–42) × (3.5–)4–5(–6) μm ($\stackrel{-}{\mathrm{x}}$ = 37 × 4.5 μm, n = 30), overlapping, 2–3-seriate, initially hyaline, becoming pale yellowish at maturity, guttulate, lunate to long fusiform, straight or slightly curved, with six transverse septa at maturity, often constricted at medium septum, widest at the middle, rounded or slightly pointed at both ends, without a mucilaginous sheath. Asexual morph: Undetermined.

Culture characteristics: Ascospores germinated on PDA within 20 h, and germ tube initially produced from both ends of the ascospores. Colonies on PDA reaching 20 mm at four weeks at room temperature, irregular, flat, centre is slightly raised, panniform, mycelium grows on the surface of PDA, white from above, brown in the centre gradually pale yellow towards the edges from below. Asexual spores and sexual spores were not formed on PDA within 60 days.

Material examined: China, Yunnan Province, Zhaotong City, Daguan County, grassland (27°44′23″ N, 103°47′59″ E), on a decaying stalk of herbaceous plant, 21 August 2021, Ying Gao, ZG17A (HKAS 124668, holotype), ex-type, CGMCC 3.23746. ibid., ZG17 (HKAS 127124, paratype), ex-paratype, CGMCC 3.23747.

Notes: Plenodomus zhaotongensis is introduced as a new species based on its distinct morphology and phylogenetic analysis of combined SSU, LSU, ITS, tub2, and rpb2 sequence data. Plenodomus zhaotongensis is closely related to Pl. agnitus strains with 91% ML and 1.00 BYPP statistical support (Figure 1). A pairwise nucleotide comparison showed that Pl. zhaotongensis differs from Pl. agnitus (CBS 121.89) in 12/529 bp of ITS (2.27%, without gaps), 12/341 bp of tub2 (3.52%, without gaps), and 18/766 bp of rpb2 (2.35%, without gaps). Plenodomus zhaotongensis differs from Pl. agnitus (sexual morph: Leptosphaeria agnita (Desm.) Ces. & De Not., Comm. Soc. Crittog. Ital. 1: 236. 1863.) in its larger ascospores (34–42 × 3.5–6 μm, lunate to long fusiform vs. 31–35 × 4–5 μm, narrowly subcylindrical) [13,55]. Therefore, based on the guidelines for new species boundaries delimitation [51,52], we introduce Pl. zhaotongensis as a novel taxon.

4. Discussion

In this study, we introduce four new species, viz. L. yunnanensis, L. zhaotongensis, Pa. kunmingensis, and Pl. zhaotongensis, associated with grasses from Zhaotong and Kunming in Yunnan Province, southwestern China, based on polyphasic approaches [56,57] through multilocus analyses of five gene loci (SSU, LSU, ITS, tub2, and rpb2) and their morphological characteristics. Although Leptosphaeria is a speciose genus with 1682 species epithets, many are likely to belong to other genera [2] and need recollecting and sequencing. However, in speciose genera, many novel taxa can still be found [58], as in this study.

Paraleptosphaeria and Leptosphaeria have similar morphologies. In our study, Pa. kunmingensis fits within the generic concept of Paraleptosphaeria and is phylogenetically closely related to Pa. macrospora (Figure 1), whereas L. yunnanensis and L. zhaotongensis clustered distinctly in Leptosphaeria (Figure 1). All species differ in morphology (Figure 2, Figure 3 and Figure 5, Supplementary Table S2). Using molecular data, Piątek et al. [20] also showed that Paraleptosphaeria and Leptosphaeria are phylogenetically distinct. We also described Pl. zhaotongensis based on the morphological characteristics (Figure 5) and molecular phylogeny (Figure 1). Nevertheless, in Plenodomus, it is challenging to have well-resolved species delimitation because many species lack molecular data and detailed morphological descriptions [5,21]. Therefore, in future studies, precise morphological characteristics, molecular data, and phylogenetic analyses should be provided for all newly introduced and existing Plenodomus species.

Except for L. maculans, leptosphaeria-like taxa are diverse and widespread. However, they are mostly found in temperate regions (Supplementary Table S1, [59,60]), with 30 species reported on grasses [29]. In addition, Leptosphaeria species seem not to be host-specific, as they have been discovered on various plant families (i.e., Adoxaceae, Apiaceae, Asteraceae, Euphorbiaceae, Fabaceae, Gentianaceae, Juglandaceae, Lamiaceae, Orobanchaceae, Plantaginaceae, Rhamnaceae, and Urticaceae) [5]. Similarly, Plenodomus is widely distributed worldwide, mainly in temperate countries such as China, Greece, France, Japan, the Netherlands, and Spain (Supplementary Table S3, [6,61]), with four species, viz. Pl. acutus, Pl. changchunensis, Pl. enteroleucus, and Pl. sorghi, are associated with grasses [6,13,62,63]. This indicates that investigations of new host plants, especially those inhabiting decomposing litter [64,65], and unstudied environments will result in undescribed taxa in this and other families, contributing to the descriptive fungal curve [66,67].

The four described species in this study were collected from climate-contrasting grasslands in Yunnan Province. In Zhaotong, subtropical and warm temperate zones coexist, with an annual average temperature of 12.6 °C [68,69], while Kunming has distinct wet and dry seasons [70,71]. In terms of fungi, many new fungal species have been reported in Yunnan Province in the last two decades. More than 1300 new fungal species have been described, accounting for nearly 25% of the total fungal species described in China [72]. This scenario is consistent with Hyde et al. [66,67], who stated that continued exploration of new environments would result in undescribed taxa.

In Yunnan’s grasslands, many grass species are the primary source of carbohydrates and feed for livestock, and fungi play a pivotal role in maintaining and shaping grass communities. Each grass-associated fungal community is responsible for specific ecological properties of the environment [29]. Our study fills some gaps in the research on Leptosphaeriaceae species in grasslands by providing detailed information on four new species from China and insights into the number of grass-associated Leptosphaeria. In addition, fungi have never been reported in Zhaotong City; thus, future studies are needed to reveal the actual fungal diversity, especially those associated with grasses.

In addition, we compared our Leptosphaeria zhaotongensis with known species that have no molecular data in Shoemaker [73]. Leptosphaeria zhaotongensis is similar to L. galii, L. raphani, L. russellii, L. stellariae, and L. byssincola in ascomata, asci and ascospores. However, they differ from Leptosphaeria galii in ascospores (guttulate vs. without guttulate) and asci (84–118 × 7.7–11 μm, vs. 45–65 × 6–8 μm); Leptosphaeria zhaotongensis differs from Leptosphaeria raphani in ascospores (guttulate vs. without guttulate), asci (84–118 μm, vs. 60–80 μm), and ascomata (380–550 × 185–300 μm, vs. 200–280 × 200–280 μm); Leptosphaeria zhaotongensis differs from Leptosphaeria russellii in asci (84–118 μm, vs. 70–85 μm) and ascomata (380–550 × 185–300 μm, vs. 200–250 × 100–150 μm); Leptosphaeria zhaotongensis differs from Leptosphaeria stellariae in ascospores (16–21 μm × 4.5–6.5 μm, guttulate, rough, vs. 26–30 × 6–7 μm, without guttulate, smooth), asci (84–118 μm, vs. 80–180 μm), and ascomata (380–550 × 185–300 μm, vs. 140–190 × 90–110 μm); and Leptosphaeria zhaotongensis differs from Leptosphaeria byssincola in asci (84–118 × 8–11 μm, vs. 80–90 × 11–13 μm). There are also obvious differences in morphology between Leptosphaeria zhaotongensis and unsequenced Leptosphaeria species.