Molecular and morphological characterization of nine new Gnomoniopsis species (Diaporthales, Gnomoniaceae): Genomic insights into evolution and phylogeny

Abstract

Gnomoniopsis comprises parasitic, endophytic, and saprophytic fungi. Since its establishment, this genus has presented considerable taxonomic challenges owing to extensive morphological overlap among species, rendering traditional classification systems that rely predominantly on phenotypic characteristics no longer applicable. To resolve the current taxonomic confusion and clarify the species diversity, phylogenetic relationships, and genomic adaptive characteristics of Gnomoniopsis, the present study was carried out. In this study, the internal transcriptional spacer (ITS), translation extension factor 1-α (tef1), and partial beta-tubulin (tub2) were analyzed and combined to observe morphological characteristics of Gnomoniopsis. The isolates were identified from chestnut (Castanea), Embelia vestita, tea (Camellia sinensis), and other hosts in Sichuan Province, as well as from Quercus species and partial leaf rot from Fujian Province. Nine new species, namely G. xiaoheijiangensissp. nov., G. embeliaesp. nov., G. yunvfengensissp. nov., G. quercussp. nov., G. ledongensissp. nov., G. yonganensissp. nov., G. conghuaensissp. nov., G. loropetalisp. nov., and G. qixiensissp. nov., were obtained. Phylogenomic analysis based on 7,942 orthologous proteins’ clusters revealed distinct species boundaries within Gnomoniopsis. Phylogenomics has thus effectively resolved the ambiguous phylogenetic relationships in this genus. This work identifies nine previously undescribed Gnomoniopsis species, clarifies the species diversity and conserved genomic characteristics within the genus, and lays a foundation for subsequent taxonomic analyses of Gnomoniopsis.

Introduction

The taxonomic study of Gnomoniopsis originated from early morphological investigations (Sogonov et al. 2008). Initially, Berlese treated it as a subgenus of Gnomonia based on septate ascospore features. Later, owing to the poor taxonomic stability of these morphological traits, this group was temporarily merged into Gnomonia. The year 2010 represented a crucial turning point for the systematics of this genus. Based on molecular phylogenetic analyses of ITS, tub2 and tef1, Walker et al. (2010) confirmed its monophyly, established it as a distinct genus, proposed new species and new combinations, and compiled an identification key for 13 species, which laid a foundation for its systematic study (ME 1978; Walker et al. 2010). Subsequently, relevant studies expanded globally, and it was confirmed that Gnomoniopsis mainly colonizes host plants of Fagaceae, Rosaceae and other plant families. Since 2018, research on Gnomoniopsis in China has accelerated (Walker et al. 2010; Jiang and Tian 2019; Yang et al. 2020; Jiang et al. 2021b). Several studies integrated morphological and molecular phylogenetic evidence to discover multiple new species across several Chinese provinces, greatly enriching the domestic species record and advancing the taxonomic and distribution research of this genus in East Asia.

Gnomoniopsis comprises parasitic, endophytic, and saprophytic fungi within Gnomoniaceae, Diaporthales, Sordariomycetes, Ascomycota. Gnomoniopsis is widely distributed throughout the world and mainly colonizes plant tissues such as Fagaceae, Salicaceae and Rosaceae. (Voglmayr et al. 2012, 2017). To date, 54 species of Gnomoniopsis have been recorded in Index Fungorum (http://www.indexfungorum.org/, accessed on 25 March 2026), among which the most typical pathogens were G. clavulata (Sogonov et al. 2008), G. smithogilvyi (Crous et al. 2012), G. daii (Jiang and Tian 2019) and G. chinensis (Jiang et al. 2020). These pathogens can cause plant disasters such as white oak (Quercus alba) and chestnut (C. mollissima) in North America, causing great losses to the local economy (Yang et al. 2020; Jiang et al. 2021a).

The mycelium of Gnomoniopsis typically grows within plant tissues, is septate, hyaline or pale in color (Zhang et al. 2025b). The sexual stage produces the ascomata, which is generally buried in the host tissue or semi-buried, has an ostiole, is usually spherical or subspherical, and varies in color from light to dark. The ascus is clavate or cylindrical and contains multiple ascus spores, which are generally single-celled, colorless, or pale, oval to fusiform, and vary in size and morphology from species to species (Walker et al. 2010; Wang et al. 2022). The asexual stage can produce conidia. Conidia are morphologically variable, ranging from cylindrical to ellipsoid. Conidia are borne on conidiophores, which are solitary or fasciculate (Jiang et al. 2022).

Breakthroughs in modern sequencing approaches now enable genomics to effectively trace fungal evolutionary lineages and uncover how pathogens trigger infection processes (Farrer et al. 2017; Wang et al. 2023b; Ruan et al. 2024; Mondo and Grigoriev 2025; Naqvi et al. 2025; Zhang et al. 2025a). Functional genomic features, including carbohydrate-active enzymes (CAZymes) and secondary metabolite biosynthetic gene clusters (BGCs), are key genetic components that determine fungal host adaptation, cell wall degradation capacity, and pathogenic potential (Cantarel et al. 2009; Keller 2019). Nevertheless, genomic data focusing on the fungal genus Gnomoniopsis remain extremely scarce. As of December 7, 2025, only four described species in this genus have publicly available whole- genome sequences in the NCBI database. The lack of sufficient genomic resources severely limits our understanding of interspecific evolutionary divergence and functional differentiation within Gnomoniopsis. Given the unresolved taxonomic ambiguity, insufficient species investigation, and limited genomic information for Gnomoniopsis, the present study was conducted to explore the species diversity, phylogenetic relationships, and functional genomic characteristics of endemic Gnomoniopsis fungi in China through integrated morphological, multi-locus phylogenetic, and comparative genomic analyses.

Materials and methods

Sample processing and morphological analysis

To broaden sampling coverage and obtain diverse plant materials, diseased and decaying leaf samples were collected in 2025 from forested areas of Yunnan, Hainan, Fujian, Northeast China, and other provinces across China. This study included fungal strains isolated from two types of plant materials: fresh symptomatic leaf tissues with active necrotic lesions and naturally decaying senescent leaves. Isolates obtained from symptomatic materials were collected from fresh, pathogen-damaged leaf tissues with typical necrotic spots and lesion browning, representing parasitic or pathogenic fungal populations. Other strains were isolated from naturally senescent and decaying leaves collected in the field, which represent saprophytic or endophytic fungal resources colonizing dead plant tissues. Completely decomposed fragments with ambiguous symptoms were excluded during sampling to ensure accurate isolation sources. From these collections, nine new fungal species described in the present study were isolated and identified from lesions on Embelia vestita, Lindera communis Hemsl., Quercus griffithii and Castanopsis chinensis. Lesion samples (5 mm × 5 mm) were cut from diseased leaves with scissors. The samples were soaked in 75% ethanol for 1 minute, then rinsed with sterile water for 30 seconds, followed by immersion in 5% hypochlorous acid solution for 1 minute, and finally rinsed with sterile water three times. After cleaning, the samples were placed on sterilized clean filter paper to absorb moisture, and then transferred to the surface of potato dextrose agar (PDA) medium (composed of 200 g potato, 20 g agar powder, 20 g glucose, 1000 mL distilled water, with pH adjusted to 7.0). After marking the date and sample number, the medium was incubated in a dark environment at 25°C for 3–5 days. The growth of hyphae around the leaf samples was observed regularly; once a certain number of hyphae appeared around the small leaf fragments the outgrowing marginal hyphae with no visible contamination were selected, a 5 mm × 5 mm agar block with hyphae was cut using an inoculating needle and transferred to a new PDA plate for further cultivation to obtain pure cultures. Single-hypha purification was repeated 2–3 times until axenic cultures without contaminant fungi or bacteria were confirmed. On the 7^th, 14^th, and 21^st days, images of the front and back sides of the colonies were captured using a digital camera (Canon PowerShot G7X; Canon Inc., Beijing, China). A stereomicroscope (Olympus SZX10; Olympus Optical Co., Ltd., Beijing, China) and an optical microscope (Olympus BX53; Olympus Optical Co., Ltd., Japan) were used to observe the growth status of hyphae and sporulation. A high-definition digital camera (Olympus DP80; Olympus Optical Co., Ltd., Tokyo, Japan) was employed to photograph fungal structures such as conidiophores, conidial structures, and conidia. The microscopic structures of each strain were measured using Digimizer, with 20–30 samples measured for each structure. All strains were preserved in a 10% sterile glycerol solution and stored at 4°C. The type specimens were deposited in the Herbarium of Shandong Agricultural University (SAUCC). Voucher specimens were deposited in the Herbarium of the Department of Plant Pathology, Shandong Agricultural University (HSAUP, Taian, China), and the Herbarium Mycologicum Academiae Sinicae (HMAS, Beijing, China) of the Institute of Microbiology, Chinese Academy of Sciences. The living type cultures were deposited in the Culture Collection Center of Shandong Agricultural University (SAUCC).

DNA extraction and sequencing

Genomic DNA of the strains grown on PDA plates was extracted using the CTAB (cetyltrimethylammonium bromide) method. When the hyphae grew adequately on PDA plates, 0.2 g of hyphae was scraped off with a sterile scalpel under aseptic conditions and transferred to a 1.5 mL centrifuge tube. Pre-warmed CTAB buffer was added to the tube, followed by homogenization of the hyphae using the steel bead grinding method. After complete homogenization, the centrifuge tube containing the lysate was placed in a 65°C water bath for 2 hours to facilitate nucleic acid release and increase cell membrane permeability. A centrifuge was used to separate the aqueous phase (4°C, 12,000 rpm, 10 minutes). The clear supernatant was transferred to a new sterile centrifuge tube, and an equal volume of chloroform-isoamyl alcohol mixture (24:1) was added to induce phase separation, allowing genomic DNA to be selectively partitioned into the aqueous layer (Wang et al. 2023a; Zhang et al. 2025b). After thorough vortexing, a second centrifugation was performed. Subsequently, the upper aqueous phase was collected and subjected to another round of chloroform-isoamyl alcohol extraction to improve DNA purity, preparing for the subsequent precipitation step.

In this study, the extracted genomic DNA was amplified via polymerase chain re-action (PCR) using the ITS, tef1, and tub2 genes (see Table 1). The primers used were as follows: ITS: ITS4/ITS5; tef1: EF1-728F/EF2; tub2: Bt-2a/Bt-2b. The PCR amplification system had a total volume of 25 µL, which contained 9.5 µL of double-distilled water (ddH₂O), 12.5 µL of 2×Taq Plus Master Mix (with dye, purchased from Yeasen Bio-technology Co., Ltd., Shanghai, China; product No.: 10154ES03), 1 µL of forward and reverse primers, and 1 µL of the strain DNA extract. The PCR amplification products were detected by 2% agarose gel electrophoresis, and the amplification effect was observed under an ultraviolet lamp. Subsequent primer synthesis and gene sequencing were entrusted to Sangon Biotech (Shanghai) Co., Ltd. After receiving the sequencing results, MEGA v.7.0 software was used for sequence analysis, comparison, and assembly. The processed sequences were compared using BLAST (http://blast.ncbi.nlm.nih.gov/). The sequences of the five taxa introduced in this study, obtained after comparison, have been uploaded to and deposited in GenBank of the National Center for Biotechnology Information (NCBI, USA). The GenBank accessions referenced in this study were collated and summarized in the Table 2.

Table 1.

Molecular markers and their PCR primers and programs used in this study.

Loci	PCR Primers	Sequence (5’—3’)	PCR Cycles	References
ITS	ITS4	TCC TCC GCT TAT TGA TAT GC	(95°C: 30 s, 55°C: 30 s, 72°C: 1 min) ×35 cycles	White et al. (1990)
	ITS5	GGA AGT AAA AGT CGT AAC AAG G
tef1	EF1-728F	CAT CGA GAA GTT CGA GAA GG	(95°C: 30 s, 48°C: 30 s, 72°C: 1 min) ×35 cycles	Carbone and Kohn (1999)
	EF2	GGA RGT ACC AGT SAT CAT GTT		O’Donnell et al. (1998)
tub2	Bt-2a	GGT AAC CAA ATC GGT GCT GCT TTC	(95°C: 30 s, 53°C: 30 s, 72°C: 1 min) ×35 cycles	Glass and Donaldson (1995)
	Bt-2b	ACC CTC AGT GTA GTG ACC CTT GGC

Table 2.

GenBank numbers used in the phylogenetic analysis of Gnomoniopsis.

Taxon	Strain	GenBank Accession Numbers
		ITS	tef1	tub2	References
Apiognomonia errabunda	AR 2813	DQ313525	DQ313565	DQ862014	Sogonov et al. (2007)
Gnomoniopsis alderdunensis	CBS 125679	GU320826	GU320813	GU320788	Walker et al. (2010)
G. alderdunensis	CBS 125680*	GU320825	GU320801	GU320787	Walker et al. (2010)
G. alderdunensis	CBS 125681	GU320827	GU320802	GU320789	Walker et al. (2010)
G. angolensis	CBS 145057*	MK047428	NA	NA	Crous et al. (2018)
G. annonae	CGMCC3.28195*	PQ325623	PQ336654	PQ336678	Zhang et al. (2025b)
G. annonae	SAUCC6333	PQ325624	PQ336655	PQ336679	Zhang et al. (2025b)
G. annonae	CGMCC3.28194	PQ325625	PQ336656	PQ336680	Zhang et al. (2025b)
G. annonae	SAUCC6423	PQ325626	PQ336657	PQ336681	Zhang et al. (2025b)
G. castanopsidis	CFCC 54437*	MZ902909	MZ936385	NA	Jiang et al. (2021b)
G. castanopsidis	CFCC 55878	MZ902910	MZ936386	NA	Jiang et al. (2021b)
G. chamaemori	CBS 804.79	GU320817	GU320809	GU320777	Walker et al. (2010)
G. chinensis	CFCC 52286*	MG866032	MH545370	MH545366	Jiang et al. (2020)
G. chinensis	CFCC 52287	MG866033	MH545371	MH545367	Jiang et al. (2020)
G. chinensis	CFCC 52288	MG866034	MH545372	MH545368	Jiang et al. (2020)
G. chinensis	CFCC 52289	MG866035	MH545373	MH545369	Jiang et al. (2020)
G. clavulata	CBS 121255	EU254818	GU320807	EU219211	Sogonov et al. (2008)
G. comari	CBS 806.79	EU254821	GU320810	EU219156	Sogonov et al. (2008)
G. comari	CBS 807.79	EU254822	GU320814	GU320779	Sogonov et al. (2008)
G. comari	CBS 809.79	EU254823	GU320794	GU320778	Sogonov et al. (2008)
G. conghuaensis	SAUCC1614603*	PZ169404	PZ191632	PZ189102	This study
G. conghuaensis	SAUCC1614604	PZ169405	PZ191633	PZ189103	This study
G. daii	CFCC 54043*	MN598671	MN605519	MN605517	Jiang and Tian (2019)
G. daii	CMF002B	MN598672	MN605520	MN605518	Jiang and Tian (2019)
G. daii	CFCC 55517	MZ902911	MZ936387	MZ936403	Jiang and Tian (2019)
G. daii	CFCC 55294B	MZ902912	MZ936388	MZ936404	Jiang and Tian (2019)
G. diaoluoshanensis	SAUCC DL0963*	ON753744	ON759769	ON759777	Wang et al. (2022)
G. diaoluoshanensis	SAUCC DL0964	ON753743	ON759768	ON759776	Wang et al. (2022)
G. embeliae	SAUCC925701*	PV134370	PZ207028	PX409985	This study
G. embeliae	SAUCC925702	PV134371	PZ207029	PX409986	This study
G. euryae	CGMCC3.28234*	PQ325629	PQ336660	PQ336684	Zhang et al. (2025b)
G. euryae	SAUCC3977	PQ325630	PQ336661	PQ336685	Zhang et al. (2025b)
G. fagacearum	CFCC 54316*	MZ902916	MZ936392	MZ936408	Jiang et al. (2021b)
G. flava	CFCC 71563*	PV257808	PV268106	PV339811	Li et al. (2025)
G. flava	CFCC 71566	PV257809	PV268107	PV339812	Li et al. (2025)
G. flava	CFCC 71567	PV257810	PV268108	PV339813	Li et al. (2025)
G. fragariae	CBS 121226	EU254824	GU320792	EU219144	Udayanga et al. (2021)
G. fragariae	CBS 208.34	EU254826	GU320808	EU219149	Udayanga et al. (2021)
G. fragariae	CBS 125671	GU320816	GU320793	GU320776	Udayanga et al. (2021)
G. fujianensis	CGMCC3.28229*	PQ325641	PQ336672	PQ336696	Zhang et al. (2025b)
G. fujianensis	SAUCC1260	PQ325642	PQ336673	PQ336697	Zhang et al. (2025b)
G. guangdongensis	CFCC 54443*	MZ902918	MZ936394	MZ936410	Jiang et al. (2021b)
G. guangdongensis	CFCC 54331	MZ902919	MZ936395	MZ936411	Jiang et al. (2021b)
G. guangdongensis	CFCC 54282	MZ902920	MZ936396	MZ936412	Jiang et al. (2021b)
G. guttulata	MS 0312	EU254812	NA	NA	Walker et al. (2010)
G. hainanensis	CFCC 54376*	MZ902921	MZ936397	MZ936413	Jiang et al. (2021b)
G. hainanensis	CFCC 55877	MZ902922	MZ936398	MZ936414	Jiang et al. (2021b)
G. idaeicola	CBS 125672	GU320823	GU320797	GU320781	Walker et al. (2010)
G. idaeicola	CBS 125673	GU320824	GU320798	GU320782	Walker et al. (2010)
G. idaeicola	CBS 125674	GU320820	GU320796	GU320780	Walker et al. (2010)
G. idaeicola	CBS 125675	GU320822	GU320799	GU320783	Walker et al. (2010)
G. idaeicola	CBS 125676	GU320821	GU320811	GU320784	Walker et al. (2010)
G. juglandis	CGMCC3.28233*	PQ325637	PQ336668	PQ336692	Zhang et al. (2025b)
G. juglandis	SAUCC2375	PQ325638	PQ336669	PQ336693	Zhang et al. (2025b)
G. lanceolata	CGMCC3.28232*	PQ325639	PQ336670	PQ336694	Zhang et al. (2025b)
G. lanceolata	SAUCC1277	PQ325640	PQ336671	PQ336695	Zhang et al. (2025b)
G. ledongensis	SAUCC1528003*	PX353467	PZ207036	PZ195445	This study
G. ledongensis	SAUCC1528004	PX353468	PZ207037	PZ195446	This study
G. lithocarpi	SAUCC YN0743*	ON753749	ON759765	ON759783	Wang et al. (2022)
G. lithocarpi	SAUCC YN0742	ON753750	ON759764	ON759782	Wang et al. (2022)
G. loropetali	SAUCC1994101*	PZ169406	PZ191634	PZ189104	This study
G. loropetali	SAUCC1994102	PZ169407	PZ191635	PZ189105	This study
G. luodingensis	CGMCC 3.29462*	PX611876	PX655142	NA	Guo et al. (2026)
G. luodingensis	GXCM RG119	PX611877	PX655141	NA	Guo et al. (2026)
G. macounii	CBS 121468	EU254762	GU320804	EU219126	Sogonov et al. (2008)
G. melastomatis	CGMCC3.28231*	PQ325631	PQ336662	PQ336686	Zhang et al. (2025b)
G. melastomatis	SAUCC0031	PQ325632	PQ336663	PQ336687	Zhang et al. (2025b)
G. melastomatis	SAUCC3040	PQ325635	PQ336666	PQ336690	Zhang et al. (2025b)
G. melastomatis	SAUCC0051	PQ325636	PQ336667	PQ336691	Zhang et al. (2025b)
G. mengyinensis	SAUCC MY0293*	ON753741	ON759766	ON759774	Wang et al. (2022)
G. mengyinensis	SAUCC MY0296	ON753742	ON759767	ON759775	Wang et al. (2022)
G. occulta	CBS 125677	GU320828	GU320812	GU320785	Walker et al. (2010)
G. occulta	CBS 125678	GU320829	GU320800	GU320786	Walker et al. (2010)
G. paraclavulata	CBS 123202	GU320830	GU320815	GU320775	Sogonov et al. (2008)
G. qixiensis	SAUCC1995302*	PZ169408	PZ191636	PZ189106	This study
G. qixiensis	SAUCC1995303	PZ169409	PZ191637	PZ189107	This study
G. quercus	SAUCC1204202a*	PV134366	PZ207034	PZ195443	This study
G. quercus	SAUCC1204202b	PV134367	PZ207035	PZ195444	This study
G. racemula	CBS 121469*	EU254841	GU320803	EU219125	Sogonov et al. (2008)
G. rosae	CBS 145 085*	MK047451	NA	NA	Crous et al. (2018)
G. rosae	CFCC 57557	ON564617	NA	NA	Crous et al. (2018)
G. rossmaniae	CFCC 54307*	MZ902923	MZ936399	MZ936415	Jiang et al. (2021b)
G. rossmaniae	CFCC 55876	MZ902924	MZ936400	MZ936416	Jiang et al. (2021b)
G. sanguisorbae	CBS 858.79	GU320818	GU320805	GU320790	Walker et al. (2010)
G. saprophytica	CGMCC3.28192*	PQ325633	PQ336664	PQ336688	Zhang et al. (2025b)
G. saprophytica	SAUCC3342	PQ325634	PQ336665	PQ336689	Zhang et al. (2025b)
G. silvicola	CFCC 54304	MZ902925	MZ936401	MZ936417	Jiang et al. (2021b)
G. silvicola	CFCC 54418*	MZ902926	MZ936402	MZ936418	Jiang et al. (2021b)
G. smithogilvyi	CBS 130190*	JQ910642	KR072534	JQ910639	Crous et al. (2012)
G. smithogilvyi	CBS 130189	JQ910644	KR072535	JQ910641	Crous et al. (2012)
G. smithogilvyi	CBS 130188	JQ910643	KR072536	JQ910640	Crous et al. (2012)
G. smithogilvyi	MUT 401	HM142946	KR072537	KR072532	Crous et al. (2012)
G. smithogilvyi	MUT 411	HM142948	KR072538	KR072533	Crous et al. (2012)
G. taishanensis	CGMCC3.28230*	PQ325645	PQ336676	PQ336700	Zhang et al. (2025b)
G. taishanensis	SAUCC1033	PQ325646	PQ336677	PQ336701	Zhang et al. (2025b)
G. tormentillae	CBS 904.79	EU254856	GU320795	EU219165	Sogonov et al. (2008)
G. xiaoheijiangensis	SAUCC921604*	PV134372	PZ191630	PZ189100	This study
G. xiaoheijiangensis	SAUCC921605	PV134373	PZ191631	PZ189101	This study
G. xishuangbannaensis	CGMCC3.25980*	PQ325643	PQ336674	PQ336698	Zhang et al. (2025b)
G. xishuangbannaensis	CGMCC3.25979	PQ325644	PQ336675	PQ336699	Zhang et al. (2025b)
G. xunwuensis	CFCC 53115*	MK432667	MK578141	MK578067	Yang et al. (2020)
G. xunwuensis	CFCC 53116	MK432668	MK578142	MK578068	Yang et al. (2020)
G. yonganensis	SAUCC1167501*	PV167928	PZ207032	PZ195441	This study
G. yonganensis	SAUCC1167502	PV167929	PZ207033	PZ195442	This study
G. yunnanensis	SAUCC YN1659*	ON753746	ON759771	ON759779	Wang et al. (2022)
G. yunnanensis	SAUCC YN1657	ON753747	ON759772	ON759780	Wang et al. (2022)
G. yunvfengensis	SAUCC1132501*	PV134368	PZ207030	PX412667	This study
G. yunvfengensis	SAUCC1132502	PV134369	PZ207031	PX412668	This study

Notes: New species established in this study are shown in bold. The ex-type strain is indicated using “*”. N/A: Not available.

Whole-genome sequencing and genome assembly and annotation

The strain was inoculated into PDB medium (35 g of PDB powder was weighed and dissolved in 1000 mL of distilled water, then aliquoted and autoclaved at 121 °C for 30 min; Beijing Solarbio Science & Technology Co., Ltd., Cat# P9240), and cultured in shake flasks at 25 °C and 100 rpm for 7 days. Mycelia were collected under aseptic conditions, frozen in liquid nitrogen for 5 min, and stored at −80 °C. The samples were sent to Wuhan Benagen Technology Co., Ltd. for library construction and high-throughput sequencing.

Raw data were subjected to quality control using fastp v1.0 (Chen 2025). The filtered reads were subjected to de novo assembly using SPAdes v3.15.5 with k-mers set to 21, 33, and 55 (Prjibelski et al. 2020). All data have been deposited in the National Center for Biotechnology Information (NCBI) database under the BioProject accession number PRJNA1456304; the corresponding GenBank accession numbers for the genome sequences are listed in Table 3.

Table 3.

BioSample and SRA NCBI number of the taxa used in phylogenomic analyses in this study.

Species	Strains	BioSample	SRA NCBI*	References
Discula destructiva	AS111	SAMEA118781011	ERS25271958	From NCBI
Gnomoniopsis castaneae	MUT401	SAMN31615005	NA	Turco et al. (2023)
G. castaneae	ICMP14040	SAMN31615006	NA	Turco et al. (2023)
G. castaneae	GN01	SAMN31615007	NA	Turco et al. (2023)
G. conghuaensis	SAUCC1614603	SAMN57401259	SRR38217291	This study
G. embeliae	SAUCC925701	SAMN57401254	SRR38217296	This study
G. ledongensis	SAUCC1528003	SAMN57401257	SRR38217292	This study
G. loropetali	SAUCC1994101	SAMN57401260	SRR38217290	This study
G. qixiensis	SAUCC1995302	SAMN57401261	SRR38217289	This study
G. quercus	SAUCC1204202a	SAMN57401256	SRR38217293	This study
G. smithogilvyi	IMI 355082	SAMN30184414	SRS14569010	From NCBI
Gnomoniopsis sp.	YN1632	SAMN40373142	SRS20766660	From NCBI
Gnomoniopsis sp.	YN0743	SAMN40373141	SRS20766659	From NCBI
Gnomoniopsis sp.	IMI 355080	SAMN30184413	SRS14569009	From NCBI
G. yonganensis	SAUCC1167501	SAMN57401258	SRR38217294	This study
G. yunvfengensis	SAUCC1132501	SAMN57401255	SRR38217295	This study
G. xiaoheijiangensis	SAUCC921604	SAMN57401253	SRR38217297	This study

* Species information described in this study is marked in bold. N/A: Not available.

Repetitive sequence families of the genome were annotated de novo using RepeatModeler v.2.0.5.(Flynn et al. 2020) The predicted results were combined with the RepBase and Dfam databases. Subsequently, genome-wide repetitive sequence annotation was performed using RepeatMasker v.4.1.6 (Chen 2004) with the parameters: -e rmblast -gff -xsmall -html -norna).

Ribosomal RNA (rRNA) genes were predicted by combining RNAmmer v.1.2 and HMMER v.2.0(Lagesen et al. 2007). Transfer RNA (tRNA) genes in the genome were identified using tRNAscan-SE v.2.0.(Chan et al. 2021). The Infernal software bundled with Rfam v.15.1 was employed to predict miRNA and snRNA sequences (Kalvari et al. 2018).

Protein-coding gene prediction was performed in three steps: first, de novo gene prediction was conducted using Augustus v3.3.3 (Stanke et al. 2004); second, protein-coding genes in the target genome were predicted based on protein-coding genes of homologous species using GeMoMa v1.9 (Keilwagen et al. 2016; Keilwagen et al. 2018); finally, EVidenceModeler v2.0.0 (Haas et al. 2008; Haas et al. 2011) was used to integrate the results from de novo prediction and homological based prediction.

Phylogenetic and phylogenomic analysis

Nucleic acid data of Gnomoniopsis were obtained from the National Center for Bi-otechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/, accessed 20 May 2025), and related GenBank accession codes were taken from the latest version of a relevant research paper. Multiple sequence alignment for nucleotide sequences of the new Gnomoniopsis taxa and publicly accessible reference data was done using MAFFT 7 (http://mafft.cbrc.jp/alignment/server/, accessed 21 May 2025). The concatenated multi-locus alignment was manually inspected and trimmed in MEGA to remove poorly aligned and ambiguous flanking regions; intron regions of tef1 and tub2 were fully retained, and only ambiguous terminal regions were deleted. Phylogenetic studies of the aligned data were carried out via maximum likelihood (ML) and Bayesian inference (BI) methods. Both analytical ways were run on their own after signing up on the CIPRES website. ML-based phylogenetic inference was performed with RAxML-HPC2 v.8.2.12 on XSEDE resources, adopting the GTRGAMMA nucleotide substitution model and using 1000 rapid bootstrap replicates to assess confidence. MrModeltest v.2.3 software was used to screen for the optimal evolutionary model, while BI analysis was conducted with MrBayes 3.2.7a on the XSEDE platform. The inferred phylogenetic tree was visualized and re-rooted with an outgroup using FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/, accessed 24 May 2025). The final tree was prepared with Adobe Illustrator CC 2019, where names of the new species were highlighted in red for easy visual distinction.

Prediction of secondary metabolite biosynthetic gene clusters and carbohydrate-active enzymes

The FASTA-formatted scaffold assembly and GFF-formatted Predicted_Genes file derived from second-generation whole-genome sequencing were submitted to the antiSMASH fungal web server (https://fungismash.secondarymetabolites.org); analyses were performed under default parameters, and biosynthetic gene clusters encoding secondary metabolites of Gnomoniopsis were retrieved from the generated result link, while carbohydrate-active enzymes (CAZymes) were annotated using dbCAN3 implemented via run_dbcan by combining Diamond homology search and HMM profile analysis, with predicted protein sequences as input and CAZyme domains identified following the default criteria of the dbCAN pipeline.

Split network inference and pairwise homoplasy index (PHI) test

Sequence alignments concatenated from ITS, tef1, and tub2 loci covering nine undescribed Gnomoniopsis species and closely related reference strains were imported into SplitsTree v.4.16.1 (www.splitstree.org) for recombination screening (Huson 1998; Huson and Bryant 2006). The LogDet distance transformation coupled with the NeighborNet algorithm was adopted to generate split networks for visualizing genetic differentiation among all operational taxonomic units. Meanwhile, pairwise homoplasy index (PHI) statistical tests were performed on the unified alignment matrix to identify signals of interspecific homologous recombination. All newly isolated strains obtained in this study were marked in red text within the network graph, which was exported as Suppl. material 3 to provide supporting evidence for species boundary delimitation (Quaedvlieg et al. 2014).

Pathogenicity tests

To assess the pathogenic potential of the nine undescribed Gnomoniopsis taxa, artificial inoculation trials were carried out on detached plant foliage. Given the practical obstacles to consistently obtaining sufficient intact, disease-free fresh tissues from each fungus’s respective native host, healthy mature leaves harvested from cultivated Chinese chestnut (Castanea mollissima) were selected as a universal substrate for all inoculation treatments.

All fungal strains were revitalized on potato dextrose agar (PDA) medium and cultured under constant temperature conditions (25 °C) for seven days to generate uniform mycelial mats. Intact chestnut leaves were surface disinfected using sequential ethanol and sodium hypochlorite rinses, followed by triple washes with sterile distilled water and air-drying under aseptic conditions. Sterile fine needles were used to create minor uniform wounds on the leaf epidermis. Circular mycelial plugs with a diameter of 5 mm were cut from the actively growing edge of each pure fungal colony and placed tightly onto the wounded leaf regions. For negative control groups, blank PDA plugs devoid of fungal mycelium were placed on identically scratched leaf tissues without fungal inoculum.

All treated leaf samples were placed into sealed plastic moist chambers and incubated at 25 °C with a 12 h light / 12 h dark alternating photoperiod. The progression of leaf discoloration and necrotic lesions was observed and recorded daily. To fulfill the criteria of Koch’s postulates, leaf segments bearing obvious necrotic lesions were excised at 14 days post inoculation. Tissue fragments collected at the boundary between healthy leaf tissue and lesion areas were subjected to surface sterilization and tissue isolation. Purified isolates recovered from diseased materials were identified via morphological observation and multi-locus sequencing of ITS, tef1 and tub2 loci to confirm their identity with the original inoculated strains (Li et al. 2025).

Results

Phylogenetic analyses

Phylogenetic analysis clarified the evolutionary relationships among species groups within the genus Gnomoniopsis. Multi-gene concatenated sequences were retrieved from GenBank and assembled, with Apiognomonia errabunda AR 2813 designated as the outgroup to construct the phylogenetic sequence matrix. The matrix comprised 50 isolates representing Gnomoniopsis and its allied taxa, containing a total of 2,450 sequence sites: the ITS region (sites 1–665), the tef1 region (sites 666–1620), and the tub2 region (sites 1621–2450). Among these sites, 1,191 were conserved, 241 were parsimony-uninformative, and 1,018 were parsimony-informative. MrModelTest analyses indicated that the Dirichlet base frequencies and the GTR+I+G evolutionary model should be adopted for Bayesian inference across all three partitions. Figure 1 presents the maximum likelihood (ML) phylogenetic tree with the highest score, and nodal supports are annotated with maximum likelihood bootstrap values and Bayesian posterior probabilities (marked as MLBS / BPP). A total of nine novel Gnomoniopsis isolates were incorporated into the phylogenomic analyses, and the results were consistent with the maximum likelihood (ML) tree mentioned above (Fig. 1). Based on the phylogenetic analyses, nine new taxa of the genus Gnomoniopsis were introduced in the present study for the first time, including G. xiaoheijiangensis sp. nov., G. embeliae sp. nov., G. yunvfengensis sp. nov., G. quercus sp. nov., G. ledongensis sp. nov., G. yonganensis sp. nov., G. conghua­ensis sp. nov., G. loropetali sp. nov., G. qixiensis sp. nov.

Figure 1.

Maximum likelihood inference tree based on a combined dataset of analyzed ITS, tef1, tub2 sequences. The Bayesian inference posterior probability (right, BIPP ≥ 0.80) and the maximum likelihood bootstrap value (left, MLBV ≥ 70%) are shown as MLBV/BIPP above the nodes. Those marked “*” in the tree are represented as ex-type or epitype strains. Strains isolated in this study are indicated in red. The scale bar at the bottom indicates 0.05 substitutions per site.

To clarify the phylogenetic independence of novel species in the genus Gnomoniopsis, species delimitation analyses were performed following the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) criterion, and the Pairwise Homoplasy Index (PHI) test was conducted to evaluate genetic recombination among closely related taxa. A Φw value of 0.05 was set as the critical threshold: a Φw value below 0.05 indicates significant genetic recombination between taxa, whereas values exceeding this threshold reflect effective genetic differentiation and distinct species boundaries. The PHI tests performed on the novel Gnomoniopsis species all yielded Φw values greater than 0.05, with no significant signals of genetic recombination detected. Collectively, the PHI recombination analyses verify that these novel taxa are markedly differentiated from their close relatives at both phylogenetic and evolutionary levels, and thus can be recognized as valid, independent species. All PHI test plots for the novel Gnomoniopsis species are provided in Suppl. material 3.

Phylogenomic analysis in Gnomoniopsis

In this study, we sequenced the genomes of nine strains of Gnomoniopsis, together with four published genomes of closely related species obtained from the NCBI (https://www.ncbi.nlm.nih.gov/datasets/) and JGI (https://genome.jgi.doe.gov/portal/) databases. Using Discula destructiva (AS111) as the outgroup, a maximum likelihood (ML) phylogenetic tree was constructed based on 7,942 clusters of orthologous protein-coding genes (Fig. 2a).

Figure 2.

Gnomoniopsis genome analysis. a. Phylogenetic relationships, composition of carbohydrate-active enzymes (CAZymes), and distribution of secondary metabolite biosynthetic gene clusters among nine novel Gnomoniopsis strains. b. Bar chart of homologous genes in each strain. c. UpSet plot of nine strains showing intersection counts among different strains in the form of bar charts.

The RAxML phylogenetic tree constructed from CDS sequences clearly resolved the phylogenetic relationships among strains of the genus Gnomo­niopsis. All taxa formed well-supported monophyletic clades with high bootstrap support values. The outgroup was located at the base of the tree, supporting the robustness of the phylogenetic topology. Branch lengths reflected the level of genetic divergence, indicating distinct interspecific differentiation and strong intraspecific conservation within Gnomoniopsis.

In the gene family number bar chart of the genus Gnomoniopsis (Fig. 2b), the composition of homologous genes among strains shows both commonalities and differences. Single-copy orthologous genes account for a relatively large proportion, indicating that these species in the genus Gnomoniopsis possess numerous conserved single-gene functions in the basic genetic framework. The presence of strain-specific unique genes may endow each strain with specific ecological adaptation or functional differentiation potential, such as specificity in host interaction and environmental stress.

Consistent with this evolutionary pattern, CAZyme profiles exhibited moderate interspecific variation among Gnomoniopsis strains, with total CAZyme counts ranging from 505 to 596. Glycoside hydrolases (GHs) constituted the predominant family, whereas carbohydrate-binding modules (CBMs) were completely conserved with four copies in all isolates. In total, 727 secondary-metabolite biosynthetic gene clusters (BGCs) were predicted across the nine strains, which varied from 67 to 94 per genome and were predominantly represented by T1PKS, NRPS-like, terpene and NRPS clusters. No strong correlation was detected between CAZyme gene count and BGC abundance, implying that carbohydrate-degrading capability and secondary-metabolite diversity are shaped by different selective forces. Several strain-specific BGC classes were also detected, which may further contribute to lineage-specific host-interaction and stress-adaptation traits (Fig. 2a).

Taxonomy

Gnomoniopsis conghuaensis

X.S. Wang, Y. Jiang, S. Wang & X.G. Zhang sp. nov.

71DC3042-847E-5B46-9E24-D0845F2B3E5C

863465

Fig. 3

Figure 3.

Gnomoniopsis conghuaensis (holotype: HMAS 354534). a. Surface of colony after 14 days on PDA; b. Reverse side of colony after 14 days on PDA; c. Colony overview; d, e. Conidiophores with attached conidia; f. Conidia. Scale bars: 10 µm (d–f).

Holotype.

CHINA • Guangdong Province, Guangzhou City, Conghua District, 23°38'26"N, 113°45'56"E, from decaying leaves, 1 March 2025, X.S. Wang, holotype HMAS 354534, ex-type living culture SAUCC1614603; paratype, ex-paratype living culture SAUCC1614604.

Etymology.

The specific epithet is named after Conghua District, the locality of its host plant collection.

Description.

The sporodochia are convex, globose or irregularly massive, mostly solitary and rarely gregarious, white in color. After incubation at 25 °C in darkness for 14 days, abundant whitish, droplet-like, slimy conidial masses exude from the surface. Conidiophores are mostly cylindrical, tapering from the base to the apex, hyaline and smooth-walled, densely arranged with immature conidia at the tip; dimensions 10.4–22.0 × 1.2–2.6 µm (mean = 16.2 × 2.0 µm, L/W = 4.0–18.3 µm, n = 20). Conidia are fusiform, hyaline, smooth, slightly curved in part, measuring 5.6–8.1 × 2.0–3.4 µm (mean = 6.8 × 2.6 µm, L/W = 1.6–4.1 µm, n = 25). Sexual morph was not observed.

Culture characteristics.

After incubation for 14 days in darkness at 25 °C on PDA medium, the colony diameter reaches 20.2–55.3 mm, with a growth rate of 1.4–3.9 mm per day. Colonies are flat and irregular in overall shape. The inner mycelium grows luxuriantly while the peripheral mycelium is sparse; the colony is overall off-white with pale brown margins. Off-white sporodochia are scattered irregularly on the colony surface.

Notes.

Phylogenetic analysis based on the combined sequences of ITS, tef1 and tub2 genes revealed that Gnomoniopsis conghuaensis is closely related to G. hainanensis (CFCC 55877) and G. paraclavulata (CBS 123202). Meanwhile, nucleotide sequence differences exist among them: the nucleotide divergences between G. conghuaensis and G. hainanensis are ITS: 45/515 (8.74%), tef1: 75/240 (31.25%), tub2: 63/364 (17.31%), and those between G. conghuaensis and G. paraclavulata are ITS: 39/518 (7.53%), tef1: 67/352 (19.03%), tub2: 54/344 (15.70%). Morphologically, the conidiophores of both species are hyaline and smooth-walled; however, those of G. hainanensis are longer than those of G. conghuaensis (16.5–26.0 × 2.5–4.5 µm, L/W = 3.7–10.4 µm vs. 10.4–22.0 × 1.2–2.6 µm, L/W = 4.0–18.3 µm). The conidia of G. hainanensis are ovate, whereas those of G. conghuaensis are fusiform. In addition, the conidia of G. hainanensis are larger than those of G. conghuaensis (8–10 × 3.4–3.9 µm, L/W = 2.1–2.9 µm vs. 5.6–8.1 × 2.0–3.4 µm, L/W = 1.6–4.1 µm).

Gnomoniopsis embeliae

X.S. Wang, Y. Jiang, S. Wang & X.G. Zhang sp. nov.

D5CBC320-501E-5740-8C35-39CC44F20F96

860858

Fig. 4

Figure 4.

Gnomoniopsis embeliae (holotype: HMAS 354062). a. Surface of colony after 14 days on PDA; b. Reverse side of colony after 14 days on PDA; c. Colony overview; d, e. Conidiophores with attached conidia; f. Conidia. Scale bars: 10 µm (d–f).

Holotype.

CHINA • Yunnan Province, Pu’er City, Ning’er Hani and Yi Autonomous County, Xiaoheijiang Forest Park, 23°12'21"N, 100°55'31"E, from diseased leaves of Castanea mollissima, 16 May 2024, X.S. Wang, holotype HMAS 354062, ex-type living culture SAUCC925701; paratype, ex-paratype living culture SAUCC925702.

Etymology.

The specific epithet “embeliae” refers to the host genus Embelia.

Description.

Sporodochium protrudes, forming spherical or blocky shapes, aggregates or solitary, is light brown in color, and its surface exudes pale yellow mucus-like conidia aggregates. Conidiophores are mostly tubular, gradually narrowing from the base towards the top. The cells are colorless and transparent, with a smooth surface, closely and orderly arranged, and gradually thinning towards the top 12.4–33.2 × 1.8–2.3 µm (mean = 22.8 × 2.0 µm, L/W = 5.4–18.4 µm, n = 20). Conidia are oval or spindle-shaped, with some individuals slightly curved, transparent and smooth 4.2–7.4 × 1.8–2.7 µm (mean = 6.1 × 2.2 µm, L/W = 1.6–4.1 µm, n = 25). Sexual morph was not observed.

Culture characteristics.

After 14 days of cultivation at 25°C in the dark, the colony on PDA reaches a diameter of 80.6–82.7 mm, with a growth rate of 5.7–5.9 mm per day. The colony is relatively flat overall: the hyphae inside the colony are sparse, while those in the outer region grow uniformly, giving the colony an overall grayish-white appearance. Light brown conidiomata are distributed in the central area of the colony.

Notes.

Phylogenetic analysis based on the combined gene sequences of ITS, tef1, and tub2 shows that Gnomoniopsis embeliae forms a distinct clade (100%/1) and is closely related to G. flava (CFCC 71563). The differences in their nucleic acid sequences are 13/552 (2.36%) for ITS, 47/550 (8.55%) for tef1, and 26/465 (5.59%) for tub2. Morphologically, the conidia of G. flava are longer than those of G. embeliae (6.5–8 × 2.5–3 µm, L/W = 2.2–3.2 µm in G. flava vs. 4.2–7.4 × 1.8–2.7 µm, L/W = 1.6–4.1 µm in G. embeliae).

Gnomoniopsis ledongensis

X.S. Wang, Y. Jiang, S. Wang & X.G. Zhang sp. nov.

2D5172C9-5B57-517B-9590-4D2DE3C37EB4

860862

Fig. 5

Figure 5.

Gnomoniopsis ledongensis (holotype: HMAS 354066). a. Surface of colony after 14 days on PDA; b. Reverse side of colony after 14 days on PDA; c. Colony overview; d, e. Conidiophores with attached conidia; f, g. Conidia. Scale bars: 10 µm (d–g).

Holotype.

CHINA • Hainan Province, Ledong Li Autonomous County, Township Road 001, 18°43'31"N, 108°50'6"E, from diseased leaves of Quercus griffithii, 4 December 2024, X.S. Wang, holotype HMAS 354066, ex-type living culture SAUCC1528003; paratype, ex-paratype living culture SAUCC1528004.

Etymology.

The specific epithet “ledongensis” is derived from Ledong Li Autonomous County, where the specimen was collected.

Description.

The sporodochia are protruding, appearing as clumps or small spheres, either solitary or clustered, and are black or light brown. When cultured in a 25°C incubator under dark conditions for 4 to 7 days, a pale yellow, viscous, cream-like aggregate of conidia exudes from the surface of the conidiomata. Conidiophores are columnar, contain septa, and taper gradually from the base to the apex, with conidia growing at the top. They are colorless and transparent, and densely arranged on the surface of the conidiomata 20.9–32.5 × 1.3–2.4 µm (mean = 26.7 × 1.8 µm, L/W = 8.7–25.0 µm, n = 20). A single conidium is fusiform or spindle-shaped, transparent and colorless 4.6–7.4 × 1.5–2.4 µm (mean = 6.1 × 1.9 µm, L/W = 1.9–4.9 µm, n = 25). Sexual morph was not observed.

Culture characteristics.

After 14 days of cultivation on PDA at 25°C in the dark, the colony diameter reaches 80.6–82.1 mm, with a growth rate of 5.7–5.9 mm per day. The colony is flat overall; most hyphae exhibit large and regular wavy growth, giving the colony an overall grayish-white appearance. Conidiomata are distributed linearly from the center to the periphery, while a small portion of hyphae grow in clumps.

Notes.

Phylogenetic analyses revealed that the newly proposed species Gnomoniopsis ledongensis in this study forms an independent clade together with G. silvicola (CFCC 54418) and G. annonae (CGMCC3.28195), indicating their close phylogenetic relationship (100%/1). However, nucleotide sequence differences exist between G. ledongensis and G. silvicola, ITS: 8/505 (1.58%); tef1: 24/298 (8.05%); tub2: 10/464 (2.16%), as well as between G. ledongensis and G. annonae, ITS: 9/587 (1.53%); tef1: 48/620 (7.74%); tub2: 14/491 (2.85%). Morphologically, there are also multiple differences among the three taxa. The conidiophores differ as follows: (20.9–32.5 × 1.3–2.4 µm, L/W = 8.7–25.0 µm vs. 7–15 × 1.5–2.5 µm, L/W = 2.8–10.0 µm vs. 7–11.5 × 1.3–2 µm, L/W = 3.5–8.8 µm). Conidia differ as: (4.6–7.4 × 1.5–2.4 µm, L/W = 1.9–4.9 µm vs. 4.5–5.3 × 2.2–2.6 µm, L/W = 1.7–2.4 µm vs. 4.6–6.8 × 1.6–2.2 µm, L/W = 2.1–4.3 µm).

Gnomoniopsis loropetali

X.S. Wang, Y. Jiang, S. Wang & X.G. Zhang sp. nov.

47DF036C-9302-5E05-81BA-258AEA9FD3C6

863466

Fig. 6

Figure 6.

Gnomoniopsis loropetali (holotype: HMAS 354535). a. Surface of colony after 14 days on PDA; b. Reverse side of colony after 14 days on PDA; c. Colony overview; d, e. Conidiophores with attached conidia; f, g. Conidia. Scale bars: 10 µm (d–g).

Holotype.

CHINA • Zhejiang Province, Quzhou City, Kaihua County, Qixi Town, 29°23'25.22"N, 118°12'35.95"E, from diseased leaves of Loropetalum chinense, 19 June 2025, X.S. Wang, holotype HMAS 354535, ex-type living culture SAUCC1994101; paratype, ex-paratype living culture SAUCC1994102.

Etymology.

The specific epithet Gnomoniopsis loropetali is derived from its host plant Loropetalum chinense.

Description.

Sporodochia are convex or immersed in the substrate, globose or irregularly massive, solitary, light brown. After incubation at 25 °C in darkness for 14 days, abundant whitish, droplet-like conidial masses (containing numerous conidia) exude explosively from the surface. Conidiophores are mostly tubular, straight or curved, tapering upwards from the base, hyaline and smooth-walled, with immature conidia densely arranged at the apex, gradually attenuate toward the tip; 8.4–22.3 × 1.3–2.1 µm (mean = 15.7 × 1.8 µm, L/W = 4.0–17.2 µm, n = 20). Conidia are mostly ovate or partially slender and linear, hyaline, smooth; 3.7–5.5 × 1.2–1.9 µm (mean = 4.3 × 1.5 µm, L/W = 1.9–4.6 µm, n = 25). Sexual morph was not observed.

Culture characteristics.

After 14 days of dark incubation at 25 °C on PDA medium, the colony diameter reaches 61.9–67.0 mm with a growth rate of 4.4–4.7 mm per day. The colonies are flat with undulate margins. The inner mycelium is sparse, while the peripheral mycelium is luxuriant. The colony is overall off-white, with pale brown sporodochia scattered irregularly on the surface.

Notes.

Phylogenetic analysis based on the combined ITS, tef1 and tub2 gene sequences showed that Gnomoniopsis loropetali formed an independent clade in the phylogenetic tree, and this clade together with G. euryae (CGMCC3.28234) and G. melastomatis (CGMCC3.28231) formed a larger clade, indicating that the three taxa are closely related. However, there were certain nucleotide sequence differences among them. The divergences between G. loropetali and G. euryae are ITS: 46/580 (7.93%), tef1: 56/355 (15.77%), tub2: 78/416 (18.75%); those between G. loropetali and G. melastomatis are ITS: 46/577 (7.97%), tef1: 57/355 (16.06%), tub2: 70/401 (17.46%). Morphologically, the conidiophores of G. loropetali were longer than those of G. euryae and G. melastomatis (8.4–22.3 × 1.3–2.1 µm, L/W = 4.0–17.2 µm vs. 11.3–15.4 × 1.5–1.8 µm, L/W = 6.3–10.3 µm vs. 15–20 × 1.8–2.3 µm, L/W = 6.5–11.1 µm). In contrast, the conidia of G. loropetali were shorter and smaller than those of G. euryae and G. melastomatis (3.7–5.5 × 1.2–1.9 µm, L/W = 1.9–4.6 µm vs. 7–9.5 × 1.8–2.2 µm, L/W = 3.2–5.3 µm vs. 8.5–11 × 2.8–3.8 µm, L/W = 2.2–3.9 µm). The conidia of G. loropetali were fusiform or ovoid, whereas those of both G. euryae and G. melastomatis were fusiform.

Gnomoniopsis qixiensis

X.S. Wang, Y. Jiang, S. Wang & X.G. Zhang sp. nov.

5C333642-4916-5E67-8D8D-9B96DE349D0E

863467

Fig. 7

Figure 7.

Gnomoniopsis qixiensis (holotype: HMAS 354536). a. Surface of colony after 14 days on PDA; b. Reverse side of colony after 14 days on PDA; c. Colony overview; d, e. Conidiophores with attached conidia; f, g. Conidia. Scale bars: 10 µm (d–g).

Holotype.

CHINA • Zhejiang Province, Quzhou City, Kaihua County, Qixi Town, 29°23'25"N, 118°12'35"E, from diseased leaves of Lindera glauca, collected on 19 June 2025 by X.S. Wang, holotype HMAS 354536, ex-type living culture SAUCC1995302; paratype, ex-paratype living culture SAUCC1995303.

Etymology.

The specific epithet “qixiensis” is derived from Qixi Town, Zhe­jiang Province, where the species was collected.

Description.

Sporodochia are convex, globose or irregularly massive, gregarious or solitary, black, exuding pale yellow droplet-like conidial masses which are viscous. Conidiophores are mostly tubular, tapering upwards from the base, hyaline and smooth-walled, with immature conidia densely arranged at the apex, gradually attenuate toward the tip; measuring 7.8–33.5 × 1.6–3.2 µm (mean = 20.6 × 2.6 µm, L/W = 2.4–20.9 µm, n = 20). Conidia are ovate or fusiform, hyaline, smooth, and some are slightly curved; measuring 5.5–7.9 × 2.5–3.3 µm (mean = 6.8 × 3.0 µm, L/W = 1.7–3.2 µm, n = 25). Sexual morph was not observed.

Culture characteristics.

After 14 days of dark incubation at 25 °C on PDA medium, the colony diameter reaches 78.0–80.5 mm, with a growth rate of 5.5–5.7 mm per day. The colonies are flat with undulate margins. The peripheral mycelium grows luxuriant; the colony is overall off-white, with black sporodochia scattered irregularly on the colony surface.

Notes.

Phylogenetic analysis based on the combined ITS, tef1, and tub2 sequences showed that Gnomoniopsis qixiensis and G. yonganensis (SAUCC1167501) form an independent clade in the phylogenetic tree and have a close phylogenetic relationship. The nucleotide differences between the two species are as follows: 32/565 (5.66%) in the ITS region, 55/330 (16.67%) in tef1, and 52/504 (10.32%) in tub2. There are significant morphological differences between them: the conidiophores of G. qixiensis are short and broad, while those of G. yonganensis are long and slender (7.8–33.5 × 1.6–3.2 µm, L/W = 2.4–20.9 µm vs. 12.6–45.9 × 0.9–2.2 µm, L/W = 5.7–51.0 µm). The conidia of G. qixiensis are ovate, whereas those of G. yonganensis are crescent-shaped.

Gnomoniopsis quercus

X.S. Wang, Y. Jiang, S. Wang & X.G. Zhang sp. nov.

65D9ACB5-413D-5ED4-A4F6-1E2AE0CA040D

860861

Fig. 8

Figure 8.

Gnomoniopsis quercus (holotype: HMAS 354065). a. Surface of colony after 14 days on PDA; b. Reverse side of colony after 14 days on PDA; c. Colony overview; d, e. Conidiophores with attached conidia; f. Conidia. Scale bars: 10 µm (d–f).

Holotype.

CHINA • Fujian Province, Nanping City, Yanping District, Mangdang Mountain, 26°41'48"N, 118°7'22"E, from diseased leaves of Quercus griffithii, 22 October 2024, X.S. Wang, holotype HMAS 354065, ex-type living culture SAUCC1204202a; paratype, ex-paratype living culture SAUCC1204202b.

Etymology.

The term “quercus” is named after its host, Quercus griffithii.

Description.

The sporodochium protrudes, forming clumpy or irregular columnar structures. They are mostly solitary, with some in clusters, and are black in color. When cultured in a 25°C incubator under dark conditions for 4 to 7 days, a pale yellow, viscous, cream-like aggregate of conidia is emitted on the surface of the sporodochium. Conidiophores are columnar, tapering gradually from the base to the apex, with conidia attached to the top. The cells are colorless and transparent, distributed on the surface of the sporocarp in a dense arrangement 14.0–36.4 × 1.5–3.4 µm (mean = 25.2 × 2.4 µm, L/W = 4.1–24.3 µm, n = 20). Conidia are fusiform or spindle-shaped, transparent and colorless, with a diameter of 6.0–6.9 × 1.8–2.3 µm (mean = 6.5 × 2.1 µm, L/W = 2.6–3.8 µm, n = 25). Sexual morph was not observed.

Culture characteristics.

After 14 days of cultivation on PDA plates at 25°C in the dark, the colony diameter is 78.4–83.5 mm, with a growth rate of 5.6–5.9 mm per day. The colony is flat; most of the hyphae grow in a regular wavy pattern, and each circle of waves is covered with white villi, giving the colony an overall grayish-white appearance. The conidiomata are distributed linearly from the center to the periphery of the colony, while a small portion of the hyphae are distributed in clumps.

Notes.

Phylogenetic analysis revealed that the newly proposed Gnomoniopsis quercus in this study forms a distinct clade (97%/1) together with G. yunnanensis (SAUCC YN1659) and G. xishuangbannaensis (CGMCC3.25980), indicating their close phylogenetic relationship; however, nucleotide sequence differences exist between G. quercus and G. yunnanensis, ITS: 4/506 (0.79%); tef1: 20/547 (3.66%); tub2: 5/464 (1.08%) as well as between G. quercus and G. xishuangbannaensis, ITS: 12/555 (2.16%); tef1: 20/636 (3.14%); tub2: 6/499 (1.20%); Morphologically, the conidiophores of G. quercus are longer than those of both G. yunnanensis and G. xishuangbannaensis (14.0–36.4 × 1.5–3.4 µm, L/W = 4.1–24.3 µm vs. 9.0–18.0 × 0.5–1.57 µm, L/W = 5.7–36.0 µm vs. 7–16 × 1.3–2 µm, L/W = 3.5–12.3 µm), the conidia of G. quercus are fusiform while those of G. yunnanensis and G. xishuangbannaensis are ovoid or cylindrical, and the conidia of G. quercus are larger than those of G. yunnanensis and G. xishuangbannaensis (6.0–6.9 × 1.8–2.3 µm, L/W = 2.6–3.8 µm vs. 4.1–5.5 × 1.3–2.0 µm, L/W = 2.1–4.2 µm vs. 5–7 × 1.6–2.6 µm, L/W = 1.9–4.4 µm); in terms of morphological characteristics, the hyphae of G. quercus grow in sparse, multilayered undulating layers from the inside out, while the hyphae of G. yunnanensis grow in an abundant petal-like spreading pattern and those of G. xishuangbannaensis are undulating and abundant.

Gnomoniopsis xiaoheijiangensis

X.S. Wang, Y. Jiang, S. Wang & X.G. Zhang sp. nov.

289952A8-476F-5897-A7E8-2ED30EEA5D19

863464

Fig. 9

Figure 9.

Gnomoniopsis xiaoheijiangensis (holotype: HMAS 354533). a. Surface of colony after 14 days on PDA; b. Reverse side of colony after 14 days on PDA; c. Colony overview; d, e. Conidiophores with attached conidia; f. Conidia. Scale bars: 10 µm (d–f).

Holotype.

CHINA • Yunnan Province, Pu’er City, Ning’er Hani and Yi Autonomous County, Xiaoheijiang Forest Park, 23°12'21"N, 100°55'31"E, from diseased leaves of Castanea mollissima, 16 May 2024, X.S. Wang, holotype HMAS 354533, ex-type living culture SAUCC921604; paratype, ex-paratype living culture SAUCC921605.

Etymology.

The specific epithet “xiaoheijiangensis” is derived from Xiaoheijiang Forest Park, the type locality where this fungal species was collected.

Description.

Sporodochia convex, globose or irregularly massive, gregarious or solitary, dark brown, exuding pale yellow droplet-shaped slimy conidial masses. Conidiophores mostly tubular, tapering upwards from the base, hyaline and smooth-walled, bearing immature conidia densely arranged at the apex, gradually attenuate toward the tip; 5.2–16.7 × 1.1–2.4 µm (mean = 10.8 × 2.1 µm, L/W = 2.2–15.2 µm, n = 20). Conidia ovate or fusiform, hyaline, smooth, slightly curved in part; 5.9–8.5 × 2.8–3.7 µm (mean = 7.2 × 3.3 µm, L/W = 1.6–3.0 µm, n = 25). Sexual morph was not observed.

Culture characteristics.

After incubation on PDA at 25 °C in darkness for 14 days, the colony diameter reaches 53.4–80.8 mm, with a growth rate of 3.8–5.7 mm per day. Colonies flat, with undulate margins. Mycelium uniformly growing, predominantly off-white; pale brown sporodochia scattered in the central area of colonies.

Notes.

Phylogenetic analyses based on concatenated ITS, tef1 and tub2 sequences revealed that Gnomoniopsis xiaoheijiangensis forms a distinct clade with G. rossmaniae (CFCC 54307), indicating a close phylogenetic relationship. The nucleotide differences between the two species are as follows: ITS: 49/507 (9.66%), tef1: 109/176 (61.93%), tub2: 44/374 (11.76%). Morphologically, the conidiophores of G. xiaoheijiangensis are smaller than those of G. rossmaniae (5.2–16.7 × 1.1–2.4 µm, L/W = 2.2–15.2 µm vs. 9.0–19.0 × 2.0–3.0 µm, L/W = 3.0–9.5 µm), and the conidia of G. xiaoheijiangensis are distinctly shorter (5.9–8.5 × 2.8–3.7 µm, L/W = 1.6–3.0 µm vs. 11.6–14.6 × 3.3–3.9 µm, L/W = 2.9–4.4 µm). In addition, G. xiaoheijiangensis produces more abundant sporodochia and exudes more conidial masses compared with G. rossmaniae.

Gnomoniopsis yonganensis

X.S. Wang, Y. Jiang, S. Wang & X.G. Zhang sp. nov.

1C42FB42-39CB-56E2-93F3-7AD284754EB2

860860

Fig. 10

Figure 10.

Gnomoniopsis yonganensis (holotype: HMAS 354064). a. Surface of colony after 14 days on PDA; b. Reverse side of colony after 14 days on PDA; c. Colony overview; d, e. Conidiophores with attached conidia; f. Conidia. Scale bars: 10 µm (d–f).

Holotype.

CHINA • Fujian Province, Sanming City, Yong’an City, Taoyuan Cave beside National Highway 205, 26°1'29"N, 117°24'55"E, from Lindera communis, 23 October 2024, X.S. Wang, holotype HMAS 354064, ex-type living culture SAUCC1167501; paratype, ex-paratype living culture SAUCC1167502.

Etymology.

The specific epithet of this species is named after Yong’an City, the locality where its host plant was collected.

Description.

The sporodochia appear as massive or spherical protrusions, distributed irregularly singly on the colony. And they are black when mature and light brown in the early stage; after being cultivated in a dark environment at 25°C for 14 days, pale yellow, viscous and cream-like conidia aggregates oozed from the surface. Conidiophores are irregular columnar, gradually narrowing from the base to the top, arranged in clusters on the surface of sporodochium. Conidia are mostly attached to the top, with some in the middle section, and have a septum inside. They are transparent and colorless 12.6–45.9 × 0.9–2.2 µm (mean = 29.2 × 1.5 µm, L/W = 5.7–51.0 µm, n = 20). Conidia are crescent-shaped, transparent and colorless, and smooth 4.8–6.0 × 1.8–2.4 µm (mean = 5.5 × 2.2 µm, L/W = 2.0–3.3 µm, n = 25). Sexual morph was not observed.

Culture characteristics.

After 14 days of cultivation in the dark condition at 25°C, the diameter of the colony on PDA reached 65.2–83.6 mm, and the growth rate was 4.6–5.9 mm per day. The colony was gray and white, with flat growth. The hyphae were short and embedded on PDA, and the sporophytes were scattered irregularly in a few numbers.

Notes.

Phylogenetic analyses based on the multi-locus sequences of ITS, tef1 and tub2 revealed that Gnomoniopsis yonganensis and G. qixiensis (SAUCC1995302) formed a distinct clade with a close phylogenetic relationship. The nucleotide differences between the two species were 32/565 (5.66%) in the ITS region, 55/330 (16.67%) in tef1, and 52/504 (10.32%) in tub2. Significant morphological differences were observed between them. The conidiophores of G. yonganensis are long and slender, whereas those of G. qixiensis are shorter and broader (12.6–45.9 × 0.9–2.2 µm, L/W = 5.7–51.0 µm vs. 7.8–33.5 × 1.6–3.2 µm, L/W = 2.4–20.9 µm). The conidia of G. yonganensis are lunate, while those of G. qixiensis are ovate.

Gnomoniopsis yunvfengensis

X.S. Wang, Y. Jiang, S. Wang & X.G. Zhang sp. nov.

D918EBAA-1D47-5502-9E17-0EB882FB98D1

860859

Fig. 11

Figure 11.

Gnomoniopsis yunvfengensis (holotype: HMAS 354063). a. Surface of colony after 14 days on PDA; b. Reverse side of colony after 14 days on PDA; c. Colony overview; d, e. Conidiophores with attached conidia; f. Conidia. Scale bars: 10 µm (d–f).

Holotype.

CHINA • Fujian Province, Nanping City, Wuyishan City, Township Road 234, Yunv Peak, 27°38'33"N, 117°57'19"E, from decaying leaves, 21 October 2024, X.S. Wang, holotype HMAS 354063, ex-type living culture SAUCC1132501; paratype, ex-paratype living culture SAUCC1132502.

Etymology.

The term “yunvfengensis” comes from the host collection site – Yunv Peak.

Description.

Sporodochium protrudes and forms blocky or spherical shapes. Most of them are solitary while some are clustered. They are light brown. After being cultivated in a dark environment at 25°C for 4 to 7 days, the surface of the carrier spores exudes yellow, viscous, cream-like secretions, which contain conidia. Conidiophores are tubular and gradually narrow from the bottom upwards. The cells are colorless and transparent, distributed on the surface of the carrier body and closely clustered 10.4–28.5 × 1.2–2.3 µm (mean = 19.4 × 1.7 µm, L/W = 4.5–23.8 µm, n = 20). Conidia are spindle-shaped, transparent and colorless 5.4–8.4 × 1.9–3.0 µm (mean = 6.6 × 2.4 µm, L/W = 1.8–4.4 µm, n = 25). Sexual morph was not observed.

Culture characteristics.

After 14 days of cultivation at 25°C in the dark, the colony reaches a diameter of 78.1–83.2 mm, with a growth rate of 5.5–5.9 mm per day. The colony is flat and exhibits irregular wavy growth from the center to the periphery, presenting an overall grayish-white color. Light brown conidiomata are irregularly distributed in the inner part of the colony.

Notes.

Phylogenetic analysis of the ITS, tef1 and tub2 sequences indicates that Gnomoniopsis yunvfengensis is closely related to G. mengyinensis (SAUCC MY0293) and G. flava (CFCC 71567). In detail, G. yunvfengensis is distinguished from G. mengyinensis by 13/510 (2.55%), 68/551 (12.34%) and 30/465 (6.45%) characters in ITS, tef1 and tub2 sequences, respectively. In the same, it is distinguished from G. flava sp. nov. by 43/551 (7.80%), 92/550 (16.73%) and 45/465 (9.68%) characters in ITS, tef1 and tub2 sequences, respectively. Morphologically, the conidiophores of G. yunvfengensis are longer than those of G. mengyinensis and G. flava (10.4–28.5 × 1.2–2.3 µm, L/W = 4.5–23.8 µm vs. 8.0–11.5 × 1.3–2.2 µm, L/W = 3.6–8.8 µm vs. 11.5–26 × 1.5–2.5 µm, L/W = 4.6–17.3 µm). G. yunvfengensis and G. flava have fewer hyphae and their colonies grow relatively irregularly, while G. mengyinensis has vigorously growing hyphae.

Pathogenicity tests

Pathogenicity inoculation experiments demonstrated (Fig. 12) that all nine novel Gnomoniopsis isolates were capable of triggering necrotic lesions on wounded Castanea mollissima (chestnut, Fagaceae) leaf tissue. This assay was designed to evaluate the general necrotrophic capacity of these fungi on Fagaceae tissue, rather than host-specific pathogenicity against their original host plants. Initial faint brown discoloration emerged around the inoculation wound site 3–4 days after treatment, and these discolored areas gradually expanded outward to form distinct water-soaked necrotic plaques as incubation time extended. No abnormal discoloration or tissue rot symptoms were detected on any leaves belonging to the blank negative control group throughout the entire observation period.

Figure 12.

Pathogenicity symptoms on detached healthy Castanea mollissima leaves inoculated with nine novel Gnomoniopsis species and negative control. a, b. Gnomoniopsis xiaoheijiangensis; c, d. G. embeliae; e, f. G. yunvfengensis; g, h. G. yonganensis; i, j. G. quercus; k, l. G. ledongensis; m, n. G. conghuaensis; o, p. G. loropetali; q, r. G. qixiensis; s, t. Negative control leaves treated with blank PDA plugs without fungal mycelium. All leaf tissues were artificially wounded before inoculation, and obvious necrotic lesions formed at the inoculation sites 3–4 days post-incubation for all nine fungal taxa; no disease lesions were observed on negative control foliage. Circles mark the positions of inoculation wounds and resultant necrotic lesions.

Fungal isolation was conducted on symptomatic leaf fragments to complete Koch’s postulate verification. Fungal colonies retrieved from lesion margins exhibited colony morphologies fully consistent with the corresponding original test strains. Sequence alignment of three standard molecular markers (ITS, tef1, tub2) further confirmed that the reisolated fungal strains shared 99%–100% sequence identity with the strains initially used for inoculation. Combined phenotypic and molecular evidence satisfied the four core requirements of Koch’s postulates under this experimental condition, verifying that all nine new Gnomoniopsis species possess the capacity to induce leaf necrotic symptoms on wounded chestnut foliage.

Discussion

Rapid development of genomic technologies has advanced traditional fungal taxonomy from qualitative morphological description to an integrated “phenotype–genotype–pan–genome landscape” paradigm (McCarthy and Fitzpatrick 2019; Xu 2020; Stengel et al. 2022). As a key genus in Diaporthales (Ascomycota), Gnomoniopsis has long suffered taxonomic ambiguities. Most species in this group are mainly endophytes and phytopathogens associated with Fagaceae, Rosaceae and related hosts plants. They display highly overlapping morphological characteristics and variable spore sizes, which renders traditional morphological identification unreliable for distinguishing closely related taxa. Although multi-locus analyses based on ITS, tef1 and tub2 have become the mainstream strategy, insufficient genetic resolution still limits the discovery of cryptic species (Rossman et al. 2007; Visentin et al. 2012; Walker 2012; Jiang et al. 2021b; Turco et al. 2023). By providing in-depth genomic data such as SNPs and gene family expansion or contraction through whole-genome sequencing, genomic research solidifies the systematic revision of Gnomoniopsis, and establishes reliable molecular criteria for species delineation (Grünwald et al. 2016; Kulik et al. 2022; Merényi et al. 2023).

After phylogenetic analyses, we identified and described nine new species of the genus Gnomoniopsis collected from five Chinese provinces, which were isolated from six different host plants: four hosts belonging to the Fagaceae, two to the genus Lindera (Lauraceae), and one to the Primulaceae. Notably, two novel species, G. yunvfengensis and G. conghuaensis, were obtained from decaying leaves without clear host information. These findings greatly extend the known host range and ecological distribution of Gnomoniopsis, and confirm that the host diversity and lifestyle plasticity of this genus are far more complex than previously documented, further implying that numerous cryptic species and unexplored host associations remain to be discovered in less-investigated plant lineages and unique microhabitats. Moreover, the newly generated genome data of the nine species significantly supplement the current genomic resources of Gnomoniopsis, providing valuable molecular evidence and a fundamental dataset for future phylogenetic reconstruction, adaptive evolution analysis, and systematic taxonomic revision of this genus.

Based on phylogenetic phylogenomic tree construction (Wisecaver et al. 2014), this study reveals the evolutionary relationships of Gnomoniopsis. These evolutionary patterns are highly consistent with well-supported conclusions from previous fungal evolutionary studies. Phylogenomic analyses showed that G. castaneae and G. smithogilvyi (IMI 355082) clustered in the same clade. Their evolutionary relationships were consistent with multi-locus phylogenetic trees and highly congruent with the phylogenetic relationships reported by Lizcano Salas et al. (Lizcano Salas et al. 2024) in their study on Fusarium. These results indicate that phylogenomics, especially based on single-copy orthologous genes, is universally applicable in resolving ambiguous species boundaries in fungi, and further clarify the taxonomic boundaries among species within the genus Gnomoniopsis (Feau et al. 2011; Gostinčar 2020; Gomez-Chavarria et al. 2024; Sarawad et al. 2025).

Conclusions

This study characterizes the genomic genetic features of Gnomoniopsis strains through whole-genome sequencing. Phylogenomic results provide supplementary evidence to previous taxonomic investigations that relied solely on a limited set of barcode loci. For future species delimitation and evolutionary analysis of this genus, analytical frameworks based on multi-locus phylogeny or concatenated phylogenomic datasets are preferable to single-gene approaches. Single-locus phylogenetic methods usually lack sufficient resolution to distinguish genetically close lineages, and may produce clustering results inconsistent with the robust species boundaries supported by morphological observation, pathogenicity verification and PHI recombination tests. The genomic resources generated in this work can serve as fundamental reference data to resolve ambiguous taxonomic relationships within Gnomoniopsis. No definitive evolutionary or species-delimitation hypothesis is put forward based solely on current genomic data; further research with expanded sampling, combined with morphological comparison and physiological experimental validation, is required to optimize the identification system for species of this genus.

Citation

Wang X, Jiang Y, Liu W, Yan X, Sheng J, Ai C, Liu X, Zhang X, Meng Z, Wang S (2026) Molecular and morphological characterization of nine new Gnomoniopsis species (Diaporthales, Gnomoniaceae): Genomic insights into evolution and phylogeny. IMA Fungus 17: e208364. https://doi.org/10.3897/imafungus.17.208364

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Artificial Intelligence (AI) use

The authors accept full responsibility for the content of the manuscript, including the disclosure of any use of AI.

No AI tools were used in the preparation of this manuscript.

Adherence to national and international regulations

All the fungal strains used in this study have been legally obtained, respecting the Convention on Biological Diversity (Rio Convention).

Funding

This research was funded by National Natural Science Foundation of China (nos. 32300011, 32500003, and 32470004).

Author contributions

X.S.Wang and Y.Jiang took charge of the drawings, DNA sequencing, and data analyses and drafted the paper; X.G.Yan, J.Y.Sheng, X.S.Wang and W.W.Liu collected samples and isolated cultures; X.G.Zhang and Z.Meng revised the paper; S.Wang, X.Y. Liu and C.C.Ai revised the paper and provided funding. All authors read and approved the final manuscript.

Author ORCIDs

X. Wang https://orcid.org/0009-0005-1127-7368

Y. Jiang https://orcid.org/0009-0003-1292-610X

W. Liu https://orcid.org/0009-0009-3477-194X

X. Yan https://orcid.org/0009-0005-6257-3490

J. Sheng https://orcid.org/0009-0003-3882-6748

C. Ai https://orcid.org/0009-0002-3934-7944

X. Liu https://orcid.org/0000-0002-8808-010X

X. Zhang https://orcid.org/0000-0001-9733-8494

Z. Meng https://orcid.org/0000-0001-9085-1324

S. Wang https://orcid.org/0000-0002-7376-7638

Data availability

All of the data that support the findings of this study are available in the main text or Supplementary Information.

Supplementary materials

Supplementary material 1

Suppl. data 1–4: the sequences after MEGA for Gnomoniopsis

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Xingsheng Wang, Yang Jiang, Wenwen Liu, Xigang Yan, Jinyan Sheng, Congcong Ai, Xiaoyong Liu, Xiuguo Zhang, Zhe Meng, Shi Wang

Data type

zip

Explanation note

suppl. data 1. The ITS sequences for Gnomoniopsis after MEGA. suppl. data 2. The tef1 sequences for Gnomoniopsis after MEGA. suppl. data 3. The tub2 sequences for Gnomoniopsis after MEGA. suppl. data 4. The ITS–tef1–tub2 sequences for Gnomoniopsis after MEGA.

Supplementary material 2

Single gene trees for Gnomoniopsis

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Xingsheng Wang, Yang Jiang, Wenwen Liu, Xigang Yan, Jinyan Sheng, Congcong Ai, Xiaoyong Liu, Xiuguo Zhang, Zhe Meng, Shi Wang

Data type

zip

Explanation note

fig. S1. Phylogenetic trees constructed using Maximum likelihood (ML) analyses based on ITS for Gnomoniopsis. fig. S2. Phylogenetic trees constructed using Maximum likelihood (ML) analyses based on tef1 for Gnomoniopsis. fig. S3. Phylogenetic trees constructed using Maximum likelihood (ML) analyses based on tub2 for Gnomoniopsis.

Supplementary material 3

PHI species delimitation test for Gnomoniopsis

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Xingsheng Wang, Yang Jiang, Wenwen Liu, Xigang Yan, Jinyan Sheng, Congcong Ai, Xiaoyong Liu, Xiuguo Zhang, Zhe Meng, Shi Wang

Data type

zip

Explanation note

figures S1–S6 exhibit pairwise homoplasy index (PHI) analytical graphs used to delimit nine novel species within Gnomoniopsis.

Supplementary material 4

Analysis of secondary metabolite biosynthetic gene clusters of Gnomoniopsis

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Xingsheng Wang, Yang Jiang, Wenwen Liu, Xigang Yan, Jinyan Sheng, Congcong Ai, Xiaoyong Liu, Xiuguo Zhang, Zhe Meng, Shi Wang

Data type

zip

Explanation note

tables S1–S9 show the detailed analyses of secondary metabolite biosynthetic gene clusters for the nine novel Gnomoniopsis species described in this study.