Five New Species of Gibellula (Hypocreales, Cordycipitaceae) from China

Abstract

The genus Gibellula (Cordycipitaceae, Hypocreales) comprises highly specialized, obligate pathogens that exclusively parasitize spiders. In this study, five new species were delimited based on morphological and phylogenetic evidence from a six-locus dataset (nrSSU, ITS, nrLSU, tef-1α, rpb1, rpb2). Specimens were collected from northeastern (Jilin and Liaoning Provinces) and southwestern (Yunnan Province) China. Phylogenetic analyses resolved these collections into five distinct, well-supported lineages, described as G. baishanensis, G. jilinensis, G. kunmingensis, G. paralongispora, and G. yunnanensis spp. nov. Among these, G. baishanensis and G. jilinensis were identified as sister taxa, whereas G. kunmingensis formed an independent lineage. Gibellula paralongispora was recovered as a sister to G. longispora, and G. yunnanensis as a sister to G. attenboroughii; both new species are supported by significant morphological distinctions (e.g., conidiophore length and conidial shape). This study provides detailed descriptions, illustrations, and morphological comparisons for these taxa, thereby enriching the taxonomy of Gibellula. Furthermore, the records from Jilin and Liaoning represent only the second documented occurrence of the genus in northeastern China, significantly expanding its known geographic range.

1. Introduction

The order Hypocreales (Sordariomycetes, Ascomycota) comprises a diverse group of fungi, many of which are entomopathogens known to infect insects and spiders [1,2]. Among these, the families Clavicipitaceae, Cordycipitaceae, Ophiocordycipitaceae, and Polycephalomycetaceae represent the most species-rich lineages of arthropod-pathogenic fungi [3,4,5,6,7]. To date, approximately 90 fungal species have been reported as pathogens of spiders (Araneae), spanning 16 families [8]. These arachnopathogenic fungi are distributed across 16 genera, including Arachnidicola, Beauveria, Clonostachys, Cordyceps, Engyodontium, Gibellula, Hevansia, Hirsutella, Hymenostilbe, Jenniferia, Lecanicillium, Ophiocordyceps, Parahevansia, Polystromomyces, Purpureocillium, and Torrubiella [9,10,11,12]. Of particular interest is the genus Gibellula, which is distinguished by its exclusive pathogenicity to spiders and its high degree of host specificity.

The genus Gibellula was established by Cavara in 1894, with G. pulchra designated as the type species [13]. This taxon is characterized by aspergillus-like conidiophores terminating in a vesicle that bears metulae, each supporting clusters of phialides which, in turn, produce chains of conidia. Despite its distinctive morphology, the taxonomic framework of Gibellula remains partially unresolved, primarily due to the absence of a clearly designated type specimen and viable cultures of G. pulchra, leading to persistent nomenclatural ambiguity [10]. Phylogenetic studies currently recognize 23 species, although molecular data are still lacking for several taxa. Sexual–asexual connections have been confirmed for only nine species; the sexual morphs typically form superficial, ovoid perithecia on loose mycelial mats, with cylindrical asci featuring a thickened apex and filiform ascospores that disarticulate into part-spores [10,14,15,16,17,18]. Most species, however, are known exclusively from their asexual morphs, which exhibit either aspergillus-like or penicillium-like conidiophores [10].

Gibellula species, commonly found on spiders inhabiting the abaxial surfaces of dicotyledonous leaves, dead stems, and other substrates, exhibit a broad global distribution across temperate, subtropical, and tropical regions, from Argentina and Brazil to China, Japan, the United States, and elsewhere [9,10]. Despite this wide distribution and the documentation of more than ten species in China to date [14,15,16,17,18,19,20], the diversity of Gibellula in East Asia remains incompletely explored, particularly in under-surveyed regions. The continual discovery of new taxa through integrated morphological and phylogenetic approaches underscores the need for further investigation.

In this study, we report five spider-pathogenic fungal isolates collected from Jilin, Liaoning and Yunnan Provinces, China. Based on comprehensive morphological examinations and multi-locus phylogenetic analyses, these isolates are proposed as five new species of Gibellula. Detailed descriptions and comparisons with morphologically and phylogenetically related taxa are provided.

2. Materials and Methods

2.1. Specimen Collection and Fungus Isolation

The fungal specimens examined in this study were mainly collected from Jilin Province, with supplementary materials obtained from Yunnan Province, China. During field surveys, specimens were photographed in situ, and relevant ecological data were documented. Samples were transported under controlled conditions (4 °C) in plastic containers and subsequently transferred to the laboratory for identification. Voucher specimens were deposited in the Herbarium of Guizhou Medical University (GMB). For fungal isolation, samples were first subjected to surface sterilization by immersion in 30% hydrogen peroxide for five minutes, followed by two rinses with sterile distilled water. Excess moisture was removed using sterile filter paper [21]. Following aseptic removal of the epidermis, tissue fragments were transferred onto potato dextrose agar (PDA) plates. Purified cultures were obtained by sub-culturing and maintained either on PDA slants at 4 °C for long-term storage or in an incubator at 25 °C for active growth [22]. Living cultures have been deposited in the Guizhou Medical University Culture Collection (GMBC).

2.2. Morphological Observations

Morphological characterization was based on asexual reproductive structures developed on the host. Observations were conducted at multiple levels, from macroscopic assessment to detailed examination using dissecting and compound microscopes. Macroscopic characterization focused on the number, colour, shape, and length of synnemata, as well as the colour of the mycelium covering the host. These characteristics were examined using a Nikon SMZ745T stereomicroscope (Tokyo, Japan). Microscopic characterization included assessment of the shape and size of vesicles, metulae, phialides, conidial heads, conidia, and conidiophores, as well as the arrangement pattern of conidiophores on the synnematal surface. Microscopic examination and image capture were performed using a Nikon ECLIPSE Ni compound microscope (Nikon, Tokyo, Japan) equipped with a Canon EOS 700D digital camera. Asexual structures such as phialides and conidia were mounted in lactophenol cotton blue solution for detailed observation. Measurements were conducted using Tarosoft (R) Image Frame Work (v.0.9.7). PDA cultures were studied for important morphological characters such as conidia and phialides.

2.3. DNA Extraction, Polymerase Chain Reaction (PCR), and Sequencing

The samples were put in a sterile centrifuge tube and processed until they were completely pulverized using sterile fine rods. The genomic DNA purification kit (Qiagen GmbH, Hilden, Germany) was used to isolate genomic DNA in accordance with the manufacturer’s instructions. The purified DNA was stored at −20 °C. The PCR mixture (25 µL) consisted of 1 µL of DNA template, 1 µL of each forward and reverse primer (10 µM each), 9.5 µL of ddH₂O, and 12.5 µL of 2× Taq PCR Master Mix (TIANGEN, Beijing, China). The primer combination NS1 and NS4 were used to amplify the nuclear ribosomal small subunit (nrSSU) [23]. The primer combination ITS4 and ITS5 were used to amplify the nuclear ribosomal internal transcribed spacer region (ITS) [23]. The primer pair 28F and 28R was used to amplify the nuclear ribosomal large subunit (nrLSU) [24]. The primer combination TEF-F and TEF-R were used to amplify the translation elongation factor 1α (tef-1α) [25,26]. The primer pairs CRPB1-5′F and CRPB1-5′R, as well as fRPB2-5F and fRPB2-7cR, were used to amplify the largest and second largest subunits of RNA polymerase II (rpb1 and rpb2) [25,27,28]. The PCR assays of the six genes were conducted as described by Wang et al. [29]. An automatic sequence analyzer (BGI Co, Ltd., Shenzhen, China) was used to sequence the PCR products after they had been separated by electrophoresis in 1.0% agarose gels and purified using the Gel Band Purification Kit (Bio Teke Co, Ltd., Beijing, China).

2.4. Phylogenetic Analyses

Sequence data for six loci (nrSSU, ITS, nrLSU, tef-1α, rpb1, and rpb2) were obtained from GenBank; relevant taxonomic information and accession numbers are provided in

Table 1. Relevant species information and GenBank accession numbers for phylogenetic research in this study.

Species	Voucher/Information	GenBank Accession Number						References
		nrSSU	ITS	nrLSU	tef-1α	rpb1	rpb2
Blackwellomyces kaihuaensis	HMAS 285455T	OQ981975	OQ981961	OQ981968	OQ980401	OQ980409	OQ980408	[35]
Blackwellomyces lateris	MFLU 18-0663T	MK086057	MK086059	MK086061	MK069471	MK084615	MK079354	[36]
Gibellula agrofloretalis	A30	PP958494	N/A	N/A	PP965288	N/A	N/A	[37]
Gibellula agrofloretalis	C11	PP958496	N/A	N/A	PP965293	N/A	N/A	[37]
Gibellula agrofloretalis	D7	PP958504	N/A	PP958435	PP965304	N/A	N/A	[37]
Gibellula attenboroughii	IMI 507230T	PQ036924	N/A	PQ036929	PQ046101	N/A	N/A	[38]
Gibellula aurea	LBMCF0003	OK329880	N/A	N/A	OK392618	N/A	OL117022	[39]
Gibellula aurea	LBMCF0006	N/A	N/A	OK329875	OK392624	N/A	OK315662	[39]
Gibellula aurea	LBMCF0007	N/A	OK329885	OK329876	OK392622	N/A	OK315663	[39]
Gibellula baishanensis	GMBC 3152T	PX425053	PX425062	PX425069	PX434402	PX434411	PX434420	This study
Gibellula baishanensis	GMBC 3153	PX425054	PX425063	PX425070	PX434403	PX434412	PX434421	This study
Gibellula brevistipitata	BCC 45580T	N/A	OK040729	OK040706	OK040697	OK040715	N/A	[7]
Gibellula cebrennini	BCC 53605T	N/A	MT477069	MT477062	MT503328	MT503321	MT503336	[40]
Gibellula cebrennini	BCC 39705	N/A	MH532874	MH394673	MH521895	MH521822	MH521859	[40]
Gibellula clavulifera var. alba	ARSEF 1915T	DQ522562	JN049837	DQ518777	DQ522360	DQ522408	DQ522467	[41,42]
Gibellula dimorpha	BCC 47518	N/A	MH532884	MH394679	MH521892	MH521819	MH521863	[7]
Gibellula flava	GNJ20200814-46	MW969660	N/A	MW969673	MW961413	MW980146	N/A	[18]
Gibellula flava	WFS20190625-25T	MW036749	N/A	MW084343	MW091325	MW384883	N/A	[18]
Gibellula fusiformispora	BCC 56802T	N/A	MT477070	MT477063	MT503329	MT503322	MT503337	[40]
Gibellula fusiformispora	BCC 45076	N/A	MH532882	N/A	N/A	MH521823	MH521860	[40]
Gibellula gamsii	BCC 27968T	N/A	MH152529	MH152539	MH152560	MH152547	N/A	[43]
Gibellula gamsii	BCC 29228	N/A	MH152533	MH152543	MH152564	MH152551	MH152558	[43]
Gibellula gamsii	EPF034	N/A	JX192720	JX192753	JX192817	N/A	N/A	[43]
Gibellula jilinensis	GMBC 3154	PX425050	PX425059	PX425066	PX434399	PX434408	PX434417	This study
Gibellula jilinensis	GMBC 3157	PX425051	PX425060	PX425067	PX434400	PX434409	PX434418	This study
Gibellula jilinensis	GMBC 3160T	PX425052	PX425061	PX425068	PX434401	PX434410	PX434419	This study
Gibellula kunmingensis	GMBC 3148T	PX425057	PX425064	PX425073	PX434406	PX434415	PX434424	This study
Gibellula kunmingensis	GMBC 3149	PX425058	PX425065	PX425074	PX434407	PX434416	PX434425	This study
Gibellula leiopus	BCC 16025	N/A	N/A	MF416548	MF416492	MF416649	N/A	[44]
Gibellula leiopus	BCC 49250	N/A	OK070780	OK070781	OK070782	OK070783	OK070784	[7]
Gibellula liaoningensis	HKAS 145357	PQ817100	PQ817098	PQ817102	PQ815114	PQ815116	PQ815118	[45]
Gibellula liaoningensis	HKAS 145358T	PQ817099	PQ817097	PQ817101	PQ815113	PQ815115	PQ815117	[45]
Gibellula longicaudata	BCC 40861	N/A	OK040730	OK040707	OK040698	OK040716	OK040724	[7]
Gibellula longispora	NHJ 12014	EU369098	N/A	N/A	EU369017	EU369055	EU369075	[46]
Gibellula longispora	GNJ20200813-16	N/A	N/A	N/A	MW961414	MW980145	N/A	[18]
Gibellula longispora	GNJ20210710-02	OL854201	N/A	OL854212	OL981628	N/A	OL981635	[18]
Gibellula mainsii	LBMCF2022.96	OQ585789	OQ589484	N/A	OQ658392	N/A	N/A	[47]
Gibellula mirabilis	LBMCF2021.70	OQ585786	OQ589481	OQ585976	OQ658389	N/A	N/A	[47]
Gibellula mirabilis	LBMCF2021.80	OQ585787	OQ589482	OQ585977	OQ658390	N/A	N/A	[47]
Gibellula mirabilis	LBMCF2022.107	OQ585792	N/A	OQ585979	OQ658395	N/A	N/A	[47]
Gibellula nigelii	NHJ 10808T	EU369099	N/A	EU369035	EU369018	EU369056	EU369076	[46]
Gibellula paralongispora	GMBC 3162T	PX425055	N/A	PX425071	PX434404	PX434413	PX434422	This study
Gibellula paralongispora	GMBC 3163	PX425056	N/A	PX425072	PX434405	PX434414	PX434423	This study
Gibellula parvula	BCC 48888	N/A	OK040731	OK040708	OK040699	OK040717	OK040725	[7]
Gibellula parvula	BCC 49748T	N/A	OK040732	OK040709	OK040700	OK040718	OK040726	[7]
Gibellula penicillioides	GNJ20200814-11	MW969650	MW969669	MW969661	MW961415	MZ215998	N/A	[18]
Gibellula penicillioides	GNJ20200814-14T	MW969651	MW969670	MW969662	MW961416	MZ215999	N/A	[18]
Gibellula penicillioides	GNJ20200814-17	MW969652	MW969671	MW969663	MW961417	N/A	N/A	[18]
Gibellula pigmentosinum	BCC 38246	N/A	MH532872	MH394672	MH521893	MH521800	MH521855	[40]
Gibellula pigmentosinum	BCC 41203T	N/A	MT477071	MT477064	MT503330	MT503323	N/A	[40]
Gibellula pilosa	BCC 57817T	N/A	OK040733	OK040710	OK040701	OK040719	N/A	[7]
Gibellula pulchra	BCC 47555	N/A	MH532885	N/A	MH521897	MH521804	N/A	[7]
Gibellula pulchra	LBMCF2020.02	OQ585783	N/A	OQ585973	OQ658386	N/A	N/A	[47]
Gibellula pulchra	LBMCF2020.03	OQ585784	N/A	OQ585974	OQ658387	N/A	N/A	[47]
Gibellula pulchra	LBMCF2020.07	OQ585785	N/A	OQ585975	OQ658388	N/A	N/A	[47]
Gibellula pulchra	LBMCF2022.GA	OQ585780	N/A	OQ585970	OQ658383	N/A	N/A	[47]
Gibellula pulchra	LBMCF2022.GB	OQ585781	OQ589487	OQ585971	OQ658384	N/A	N/A	[47]
Gibellula queenslandica	BRIP 72767aT	N/A	OR452099	OR452103	OR459912	N/A	OR459907	[48]
Gibellula scorpioides	BCC 47976T	N/A	MT477078	MT477066	MT503335	MT503325	MT503339	[40]
Gibellula solita	BCC 45574T	N/A	OK040736	OK040712	OK040703	OK040721	N/A	[7]
Gibellula sp.	NHJ 5401	EU369102	N/A	N/A	N/A	EU369059	EU369079	[46]
Gibellula sp.	NHJ 10788	EU369101	N/A	EU369036	EU369019	EU369058	EU369078	[46]
Gibellula trimorpha	BCC 36526T	N/A	OK040737	N/A	OK040704	OK040722	OK040728	[7]
Gibellula trimorpha	BCC 36538	N/A	MH532867	MH394668	MH521890	MH521817	MH521861	[7]
Gibellula unica	BCC 45112	N/A	OK040738	N/A	OK040705	OK040723	N/A	[7]
Gibellula unica	BCC 46590	N/A	MH532883	MH394678	N/A	MH521803	MH521866	[7]
Gibellula yunnanensis	GMB 3142T	PX354537	PX354531	PX354543	PX370035	PX371911	PX371921	This study
Gibellula yunnanensis	GMB 3143	PX354538	PX354532	PX354544	PX370036	PX371912	PX371922	This study
Hevansia arachnophila	NHJ 2633	N/A	MH532900	GQ249978	MH521917	MH521843	MH521884	[43]
Hevansia minula	BCC 47519T	N/A	MZ684087	MZ684002	MZ707811	MZ707826	MZ707833	[9]
Hevansia minula	BCC 47520	N/A	MZ684088	MZ684003	MZ707812	MZ707827	MZ707834	[9]
Hevansia nelumboides	TNS 16306	MF416585	N/A	N/A	MF416475	N/A	MF416438	[44]
Hevansia novoguineensis	BCC 42675	N/A	MZ684089	MZ684004	MZ707814	N/A	MZ707835	[9]
Hevansia novoguineensis	CBS 610.80T	N/A	MH532831	MH394646	MH521885	N/A	MH521844	[49]
Jenniferia cinerea	NHJ 03510T	N/A	N/A	N/A	EU369009	EU369048	EU369070	[46]
Jenniferia cinerea	BCC 2191	GQ249956	GQ250000	GQ249971	GQ250029	N/A	N/A	[43]
Jenniferia griseocinerea	BCC 42062T	N/A	MZ684091	MZ684006	MZ707815	MZ707828	MZ707837	[9]
Jenniferia griseocinerea	BCC 42063	N/A	MZ684092	MZ684007	MZ707816	MZ707829	MZ707838	[9]
Jenniferia thomisidarum	BCC 37881T	N/A	MZ684099	MZ684010	MZ707823	MZ707830	MZ707843	[9]
Jenniferia thomisidarum	BCC 37882	N/A	MZ684100	MZ684011	MZ707824	MZ707831	MZ707844	[9]

Boldface: data generated in this study; ^T: ex-type material. Institutional acronyms: ARSEF: Agricultural Research Service Collection of Entomopathogenic Fungal Cultures (culture collection); BCC: BIOTEC Culture Collection (culture collection); CBS: Westerdijk Fungal Biodiversity Institute (culture collection); GMB: Herbarium of Guizhou Medical University (herbarium); GMBC: Guizhou Medical University Culture Collection (culture collection); HKAS: Herbarium of Cryptogams, Kunming Institute of Botany, Chinese Academy of Sciences (herbarium); IMI: CABI Bioscience UK Centre (includes both cultures and herbarium specimens); NHJ: National Herbarium of Japan (herbarium); TNS: National Museum of Nature and Science (herbarium); MFLU: Mae Fah Luang University (herbarium).

. Sequence alignment was performed using MAFFT v.7 (https://mafft.cbrc.jp/alignment/server/ (accessed on 20 October 2025)) and MEGA 7.0.26 [30], with manual adjustments made where necessary. The aligned sequences were concatenated into a single dataset using MEGA 7.0.26. Phylogenetic analyses were conducted using both Maximum Likelihood (ML) and Bayesian Inference (BI) methods. For ML analysis, the GTR + FO + G model was selected as the best-fit model, and branch support was evaluated with 1000 rapid bootstrap replicates. ML analyses were performed using RAxML v.7.0.3 [31], with additional ML analysis carried out in IQ-TREE v.2.1.3, where TIM3 + F + I + G4 was identified as the optimal model based on the Bayesian Information Criterion (BIC), and node support was assessed via ultrafast bootstrapping [32]. For BI, substitution models were selected using jModelTest v.2.1.4 [33]; the GTR + I + G model was applied to nrSSU, ITS, nrLSU, and tef-1α partitions, while GTR + I was used for rpb1 and rpb2. Bayesian analysis was run for 5 million generations in MrBayes v.3.2.7a [34]. Blackwellomyces kaihuaensis (HMAS 285455) and Blackwellomyces lateris (MFLU 18-0663) were designated as outgroup taxa. Phylogenetic trees were visualized and edited using FigTree v.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree (accessed on 20 October 2025)).

3. Results

3.1. Sequencing and Phylogenetic Analyses

A six-locus dataset (nrSSU, ITS, nrLSU, tef-1α, rpb1, and rpb2) with a total length of 5696 bp (nrSSU: 1076 bp; ITS: 776 bp; nrLSU: 949 bp; tef-1α: 992 bp; rpb1: 776 bp; rpb2: 1127 bp) was assembled to elucidate the phylogenetic relationships of Gibellula and allied genera in Cordycipitaceae. The alignment included 80 fungal specimens/isolates, comprising 66 of Gibellula, six of Hevansia, six of Jenniferia, and two outgroup specimens of Blackwellomyces. Phylogenetic trees reconstructed using maximum likelihood (IQ-TREE, RAxML) and Bayesian inference exhibited highly congruent topologies, with nodal support values (IQ-TREE-BS/RAxML-BS/PP) indicated in Figure 1.

Phylogenetic analyses strongly supported the monophyly of three genera: Gibellula (100%/100%/1), Hevansia (100%/100%/1), and Jenniferia (100%/100%/1). Hevansia and Jenniferia formed a well-supported sister clade (98%/94%/1), which in turn grouped with Gibellula with full support (100%/100%/1). Within the Gibellula clade, the five proposed new species each formed well-supported independent lineages: G. kunmingensis (GMBC 3148, 3149) as a distinct terminal branch; G. yunnanensis (GMB 3142, 3143) forming a sister clade to G. attenboroughii; G. paralongispora (GMBC 3162, 3163) forming a sister clade to G. longispora; and G. baishanensis (GMBC 3152, 3153) together with G. jilinensis (GMBC 3154, 3157, 3160) forming a distinct clade without a clear sister relationship to any other known Gibellula species. The majority of recognized Gibellula species were resolved into species-specific clades with high statistical support, further validating the current taxonomic framework.

The combined molecular dataset thus provided robust phylogenetic evidence for the monophyly of Gibellula, its phylogenetic position relative to Hevansia and Jenniferia, and the recognition of five novel species. These results offer a solid foundation for taxonomic revision and evolutionary studies of Gibellula and related genera in Cordycipitaceae.

3.2. Taxonomy

Gibellula baishanensis Y. Wang & H. Chen, sp. nov.

Figure 2.

Mycobank No: 861165

Etymology. The specific epithet refers to Baishan City in Jilin Province, China, where the holotype was collected.

Type. China, Jilin Province, Baishan City, Fusong County (42.37° N, 127.43° E; alt. 775 m), on a spider on a dead stem, July 2024, Yao Wang (holotype: GMB 3152; ex-type living culture: GMBC 3152).

Description. Host surface covered by a dense white mycelial mat. Synnemata white, numerous, arising directly from the entire host body, becoming purplish-grey upon drying. Stipes are short, erect, cylindrical, resembling a narrow head. Conidiophores 69–89 × 8–11 ($\overline{\mathrm{X}}$ = 81 × 9, n = 30) μm, few, solitary, white, arising directly from the dorsal surface or legs of the host; erect, thick-walled, septate, with wider spacing between septa toward the apex; simple, with verrucose to globose ornamentation near the apex and conspicuous septal thickening. Terminal cell of conidiophores smooth, thin-walled, 18–29 ($\overline{\mathrm{X}}$ = 24, n = 30) μm long, bearing a stipe 6–8 ($\overline{\mathrm{X}}$ = 7.3, n = 30) μm long, apically swollen. Vesicles spatulate to conical, 5–11 × 3–5 ($\overline{\mathrm{X}}$ = 9.3 × 3.3, n = 30) μm, smooth. Metulae broadly ovoid to broadly ellipsoid, 5–11 × 6–7 ($\overline{\mathrm{X}}$ = 8.6 × 6.6, n = 30) μm, borne on vesicles; vesicles together with metulae and phialides forming spherical to ovoid heads, 69–89 × 34.5–44 ($\overline{\mathrm{X}}$ = 80 × 39, n = 30) μm. Phialides 10–14 × 2–3 ($\overline{\mathrm{X}}$ = 11 × 2.3, n = 30) μm, smooth, tapering, producing conidia in chains. Conidia hyaline, smooth, lacrimoid to subclavate, gradually narrowing toward the base, thin-walled, aseptate, 5.5–7 × 2–4 ($\overline{\mathrm{X}}$ = 6 × 3, n = 50) μm. Sexual morph not observed.

Culture characteristics. Colonies on PDA grow relatively rapidly at 25 °C, reaching 28–33 mm in diameter after 30 days. Mycelium initially greyish-white to cream-white, with colony margins gradually turning pale yellow. Colonies are loose superficially and compact at the central base. Sporulation not observed in culture.

Distribution. China, Jilin Province, Baishan City.

Other Material Examined. China, Jilin Province, Baishan City, Fusong County (42.46° N, 127.59° E; alt. 637 m), on a spider on a dead stem, July 2024, Yao Wang (GMB 3153; living culture: GMBC 3153).

Notes. Phylogenetically, G. baishanensis forms a highly supported clade (IQ-TREE-BS/RAxML-BS/BI-PP = 100%/100%/1) as a sister species to G. jilinensis. Morphologically, it is characterized by its large, spherical to ovoid conidial heads (69–89 × 34.5–44 µm) and medium-sized, lacrimoid to subclavate conidia (5.5–7 × 2–4 µm). The size of the conidial heads distinguishes it from species with smaller heads, such as G. attenboroughii and G. agrofloretalis, while its conidial shape separates it from species with narrowly fusiform conidia and longer conidiophores, such as G. longispora and G. penicillioides. The combined molecular and morphological evidence supports the recognition of G. baishanensis as a distinct species.

Gibellula jilinensis Y. Wang & H. Chen, sp. nov.

Figure 3.

Mycobank No: 861166

Etymology. The specific epithet refers to Jilin Province, China, where the species was collected.

Type. China, Jilin Province, Yanbian Korean Autonomous Prefecture, Dunhua County (43.98° N, 128.31° E; alt. 636 m), on a spider on a dead stem, 26 August 2025, Kun Zhang (holotype: GMB 3160; ex-type living culture: GMBC 3160).

Description. Host surface covered by a dense white mycelial mat. Synnemata white, numerous, and arise exclusively from the dorsal surface of the host. During drying, their colour transitions from white to pale yellow, then to deep yellow or brown, and finally to purplish-grey, with spores aggregating on the dorsal surface. Stipes short and stout, cylindrical. Conidiophores few, solitary, yellow to brown, erect, thick-walled, septate, with wider spacing between septa toward the apex, simple, 78–109 × 6–8 ($\overline{\mathrm{X}}$ = 86 × 7.5, n = 30) µm. Terminal cell of conidiophores smooth, thin-walled, 18–25 ($\overline{\mathrm{X}}$ = 21, n = 30) µm long, bearing a stipe 6–8 ($\overline{\mathrm{X}}$ = 7.6, n = 30) µm long, apically swollen; vesicles spatulate to conical, 14–19 × 7–9 ($\overline{\mathrm{X}}$ = 17.1 × 8.2, n = 30) µm. Metulae broadly ovoid to broadly ellipsoid, 7–10 × 5–7 ($\overline{\mathrm{X}}$ = 7.6 × 6, n = 30) µm, borne on vesicles. Phialides cylindrical to lageniform, 9–15 × 2–3 ($\overline{\mathrm{X}}$ = 13.5 × 2.2, n = 30) µm. Vesicles together with metulae and phialides forming spherical to ovoid heads, 37–59 × 28–44 ($\overline{\mathrm{X}}$ = 44 × 35, n = 30) µm. Conidia hyaline, smooth, lacrimoid to subclavate, gradually narrowing toward the base, thin-walled, aseptate, 5.5–6.5 × 2–3 ($\overline{\mathrm{X}}$ = 6 × 2.5, n = 50) µm. Sexual morph not observed.

Culture characteristics. Colonies on PDA grow slowly at 25 °C, reaching 15–19 mm in diameter after 30 days. Mycelium initially greyish-white to cream-white, with colony margins gradually turning pale yellow. The central base darkens, and colonies are compact on the surface. Sporulation not observed in culture.

Distribution. China, Jilin Province, Baishan City and Yanbian Korean Autonomous Prefecture.

Other Material Examined. China, Jilin Province, Baishan City, Fusong County (42.69° N, 127.40° E; alt. 639 m), on a spider attached to a dead stem, July 2024, Yao Wang (GMB 3154; living culture: GMBC 3154). Yanbian Korean Autonomous Prefecture, Dunhua County (43.68° N, 128.11° E; alt. 647 m), on spiders attached to dead stems, 26 August 2024, Yao Wang (GMB 3155, GMB 3156, GMB 3157; living cultures: GMBC 3155, GMBC 3156, GMBC 3157); 1 September 2024, Yao Wang (GMB 3158, GMB 3159; living cultures: GMBC 3158, GMBC 3159).

Notes. Gibellula jilinensis forms a maximally supported sister clade with G. baishanensis (IQ-TREE-BS/RAxML-BS/BI-PP = 100%/100%/1), confirming their close relationship while underscoring their distinct species status. Morphologically, G. jilinensis is distinguished by synnemata that are restricted to the host’s dorsal surface, unlike in G. baishanensis where they also occur on the legs. Furthermore, its synnemata undergo a distinct colour transition during drying, progressing from white to pale yellow, then to deep yellow or brown, and finally to purplish-grey, with spores aggregating dorsally. The species also produces notably smaller conidial heads (37–59 × 28–44 µm) and significantly larger vesicles (14–19 × 7–9 µm) than G. baishanensis. These consistent morphological differences, supported by molecular data, validate the recognition of G. jilinensis as a new species.

Gibellula kunmingensis Y. Wang & H. Chen, sp. nov.

Figure 4.

Mycobank No: 861167

Etymology. The species epithet refers to Kunming City in Yunnan Province, China, where the holotype was collected.

Type. China, Yunnan Province, Kunming City, Wild Duck Lake Forest Park (25.21° N, 102.85° E; alt. 2110 m), on a spider attached to the underside of a leaf, August 2024, Yao Wang (holotype: GMB 3148; ex-type living culture: GMBC 3148).

Description. The host surface is densely covered by a white mycelial mat. Synnemata are white, numerous, and arise directly from the entire host body. The stipes are short, erect, cylindrical, and resemble a narrow head, ca. 5 mm long. Conidiophores numerous, distinctly clustered, white, arising directly from the mycelium covering the host or along the synnemata; erect, thick-walled, septate, with wider spacing between septa toward the apex; simple, with verrucose to globose ornamentation near the apex and conspicuous septal thickening, 52–120 × 14–16 ($\overline{\mathrm{X}}$ = 88 × 15, n = 30) µm, abruptly narrowing at the terminal cell to 7–8 ($\overline{\mathrm{X}}$ = 7.5, n = 30) µm. Terminal cell of conidiophores smooth, thin-walled, 14–21 ($\overline{\mathrm{X}}$ = 18, n = 30) µm long, bearing a stipe 5–7 ($\overline{\mathrm{X}}$ = 6, n = 30) µm long, apically swollen. Vesicles spatulate to conical, 13–17 × 5–7 ($\overline{\mathrm{X}}$ = 14.5 × 6.5, n = 30) µm, smooth, bearing broadly ovoid to broadly ellipsoid metulae, 13–17 × 5–7 ($\overline{\mathrm{X}}$ = 15.5 × 6, n = 30) µm, each supporting cylindrical to lageniform phialides, 12–21 × 7–8 ($\overline{\mathrm{X}}$ = 16 × 7, n = 30) µm. Vesicles together with metulae and phialides forming spherical to ovoid heads, 36–49 × 25–35 ($\overline{\mathrm{X}}$ = 44 × 31, n = 30) µm, occasionally much reduced in size and complexity. Conidia hyaline, smooth, lacrimoid to subclavate, gradually narrowing toward the base, thin-walled, aseptate, 2–4 × 1.2–2 ($\overline{\mathrm{X}}$ = 3.2 × 1.5, n = 50) µm. Sexual morph not observed.

Culture characteristics. Colonies on PDA grow slowly at 25 °C, reaching 28–32 mm in diameter after 30 days. Mycelium initially greyish-white to cream-white, with the colony margin gradually turning pale yellow with age. Colonies are loose on the surface but compact at the base. Sporulation not observed in culture.

Distribution. Currently known only from Kunming City, Yunnan Province, China.

Other Material Examined. China, Yunnan Province, Kunming City (25.35° N, 102.58° E; alt. 1987 m), on spiders attached to dead stems, August 2025, Hui Chen (GMB 3149, GMB 3150, GMB 3151; living cultures: GMBC 3149, GMBC 3150, GMBC 3151).

Notes. Morphological comparisons between G. kunmingensis and similar species are summarized in

Table 2. Comparison of the morphological characters of G. baishangensis, G. jilinensis, G. kunmingensis, G. paralongispora and related species.

Species	Conidiophore (μm)	Metulae (μm)	Phialide (μm)	Conidia (μm)	References
Gibellula attenboroughii	Verrucose, 80–120 × 5–8	Borne on vesicle, 10–12 × 6–8	Cylindrical to narrowly clavate, 7.5–9.5 × 2.5–3.5	Hyaline, smooth, 4–6 × 1.5–2	[38]
G. baishangensis	Verrucose to globose, 69–89 × 8–11	Broadly ovoid to broadly ellipsoid, 5–11 × 6–7	Spherical to ovoid, 10–14 × 2–3	Hyaline, smooth, 5.5–7 × 2–4	This study
G. flava	Verrucose, 33.5–123.5 × 4–9.5	Obovoid to broadly obovoid, 5.5–7 × 3.5–5.5	Narrowly obovate to clavate, 5.5–7 × 1.5–2.5	Fusiform, 3–4 × 1–2	[50]
G. jilinensis	Verrucose, 78–109 × 6–8	Broadly ovoid to broadly ellipsoid, 7–10 × 5–7	Cylindrical to lageniform, 9–15 × 2–3	Hyaline, smooth, 5.5–6.5 × 2–3	This study
G. kunmingensis	Verrucose to globose, 52–120 × 14–16	Broadly ovoid to broadly ellipsoid, 13–17 × 5–7	Cylindrical to lageniform, borne on vesicles, 12–21 × 7–8	Hyaline, smooth, 2–4 × 1.2–2	This study
G. longispora	Multiseptate, minutely roughened, 159.5–290.5 × 8.5–11	Broadly obovoid, 7.5–9.5 × 6–6.5	Narrowly clavate to cylindrical, 9.5–11 × 3–3.5	Bacilliform to cylindrical, 5.5–8 × 1–1.5	[8]
G. paralongispora	Verrucose, 111–188 × 8–10	Broadly obovate to oval, 6–11 × 4–7	Clavate, 9–11 × 2–5	Narrowly fusiform, 4.5–6 × 1.5–2	This study
G. penicillioides	Penicillate, smooth, mostly biverticillate or terverticillate, 52.5–92 × 4.5–6	Obovoid to cylindrical, 13–17.5 × 3.5–5	Broadly cylindrical, 12.5–15.5 × 3–4	7.5–9 × 2.5–3.5	[18]
G. pigmentosinum	Smooth to verrucose, 97.5–170 × 7–10	Broadly obovoid, 6–8 × 4–6	Obovoid to clavate, 5.5–8 × 2–3	Obovoid with an acute Apex 3.5–5 × 1–2	[40]
G. pilosa	Minutely roughened, 151–265 × 9–11	Broadly obovoid, 9.5–11 × 7–8	Narrowly clavate to cylindrical, 7–9 × 2.5–3	Narrowly almond shaped, 3–4 × 1.5–2	[8]
G. pulchra	Verrucose, 155–170 × 7.5–10	Cylindrical, 6.2–7.5 × 5–6	Clavate, 7.5–8 × 1.5–2.5	Fusiform to fusiform ellipsoid, 3–5 × 1.5–2.5	[17]
G. solita	Verrucose, 82–146 × 7.5–9.5	Broadly obovoid, 7–7.5 × 5–6	Narrowly clavate to cylindrical, 6–7 × 2–2.5	Occasionally globose, 2–2.5 × 1–1.5	[8]
G. yunnanensis	Verrucose, 27–58 × 3–8	Broadly obovate to oval, 6.4–9.7 × 5–7	Borne on vesicle, 6.4–9.7 × 5–7	Narrowly fusiform, 6–9 × 2.2–3.1	This study

Note: Bold labels indicate the morphological data of the new species described in this study.

. Although G. kunmingensis resembles G. pulchra in the production of numerous synnemata, it is distinguished by a combination of shorter synnemata (approximately 5 mm long), spatulate to conical vesicles, and smaller conidial heads (36–49 × 25–35 µm). Furthermore, while other related species listed in Table 2 typically produce paired or multiple synnemata ranging in colour from white to brownish-white, those of G. kunmingensis are consistently numerous and milky white. Phylogenetically, G. kunmingensis forms an independent clade with moderate support (IQ-TREE-BS/RAxML-BS/BI-PP = 74/94/-). The distinct morphological characteristics, coupled with its phylogenetic isolation, support the recognition of G. kunmingensis as a new species.

Gibellula paralongispora Y. Wang & H. Chen, sp. nov.

Figure 5.

Mycobank No: 861168

Etymology. The specific epithet refers to the morphological resemblance to Gibellula longispora.

Type. China, Jilin Province, Yanbian Korean Autonomous Prefecture, Dunhua County (43.07° N, 128.01° E; alt. 637 m), on a spider attached to the underside of a leaf, 26 August 2024, Yao Wang (holotype: GMB 3162; ex-type living culture: GMBC 3162).

Description. Mycelium covering the host, white to cream, floccose, becoming light greyish brown to violaceous-brown upon drying. Synnemata multiple, cylindrical, arising from the abdomen of the host spider, cream to yellowish white. Conidiophores 111–188 × 8–10 ($\overline{\mathrm{X}}$ = 168 × 9, n = 30) μm, densely arranged, arising secondarily from hyphae loosely attached to the synnematal surface; verrucose, multiseptate, abruptly narrowing at the apex and forming a globose vesicle, 8–10.5 × 5–8 ($\overline{\mathrm{X}}$ = 9.5 × 6, n = 30) μm. Conidial heads spherical, composed of vesicles, metulae, and phialides, 28.5–40 × 25–36.5 ($\overline{\mathrm{X}}$ = 37 × 32, n = 30) μm. Metulae broadly obovate to oval, 6–11 × 4–7 ($\overline{\mathrm{X}}$ = 8 × 5.5, n = 30) μm, borne on vesicles, each bearing several clavate phialides, 9–11 × 2–5 ($\overline{\mathrm{X}}$ = 10 × 4, n = 30) μm. Conidia narrowly fusiform, 4.5–6 × 1.5–2 ($\overline{\mathrm{X}}$ = 5 × 1.8, n = 50) μm. Neither teleomorph nor granulomanus-like synanamorph observed.

Culture characteristics. Colonies on PDA grow slowly at 25 °C, reaching 18–21 mm in diameter after 30 days. Mycelium initially greyish-white to cream-white, with margins gradually turning pale yellow to brown with age. Colonies are compact both superficially and at the base. Sporulation not observed in culture.

Distribution. Currently known from Baishan City and Yanbian Korean Autonomous Prefecture in Jilin Province, and Tieling City in Liaoning Province, China.

Other Material Examined. China, Jilin Province, Baishan City, Fusong County (42°37′ N, 127°59′ E; alt. 639 m), on a spider attached to the underside of a leaf, 29 July 2025, collected by Kun Zhang (GMB 3161; living culture: GMBC 3161); Yanbian Korean Autonomous Prefecture, Dunhua County (43.10° N, 128.05° E; alt. 630 m), on spiders attached to the underside of leaves, 26 August 2025, Kun Zhang (GMB 3163, GMB 3164; living culture: GMBC 3163, GMBC 3164); Liaoning Province, Tieling City, Xifeng County (42°54′ N, 124°41′ E; alt. 346 m), on a spider attached to the underside of a leaf, 25 September 2025, collected by Kun Zhang (GMB 3165; living culture: not available).

Notes. Gibellula paralongispora forms a sister clade to G. longispora in the phylogeny, with strong support (IQ-TREE-BS/RAxML-BS/BI-PP = 97%/96%/0.99). Morphologically, both species share similar conidiophore structures and globose conidial heads. However, G. paralongispora differs in its narrower conidia (4.5–6 × 1.5–2 μm vs. 5–7 × 1–2 μm in G. longispora) and longer conidiophores (111–188 μm vs. 60–153.5 μm). These consistent morphological distinctions, together with molecular phylogenetic evidence, support the recognition of G. paralongispora as a distinct species.

Gibellula yunnanensis Y. Wang & H. Chen, sp. nov.

Figure 6.

Mycobank No: 861190

Etymology. The specific epithet refers to Yunnan Province, China, where the species was collected.

Type. China, Yunnan Province, Kunming City, Wild Duck Lake Forest Park (25.68° N, 102.47° E, alt. 2100 m), on a spider attached to the underside of a leaf, 26 August 2024, Yao Wang (holotype: GMB 3142; ex-type living culture: not available).

Description. Mycelium covering the host, white to creamy yellow, becoming light greyish-brown to violaceous-brown when dried. Synnemata multiple, cylindrical, arising from the abdomen of the host spider, cream to yellowish white. Conidiophores 27–58 × 3–8 ($\overline{\mathrm{X}}$ = 33 × 6, n = 30) μm, crowded, arising from hyphae loosely attached to the surface of the synnema, verrucose, multiseptate, suddenly narrowing to a tip, and forming a globose vesicle, 6–9 × 5.5–8 ($\overline{\mathrm{X}}$ = 7.5 × 6, n = 30) μm. Conidial heads composed of vesicle, metulae, and phialides, 22.5–31 × 36–46.5 ($\overline{\mathrm{X}}$ = 25 × 42, n = 30) μm. Metulae broadly obovate to oval, 6.4–9.7 × 5–7 ($\overline{\mathrm{X}}$ = 8 × 6, n = 30) μm, borne on vesicles. Phialides clavate, 6–8 × 2–3 ($\overline{\mathrm{X}}$ = 7 × 2.2, n = 30) μm, several per metula. Conidia narrowly fusiform, 6–9 × 2.2–3.1 ($\overline{\mathrm{X}}$ = 7.6 × 2.8, n = 50) μm. Teleomorph and granulomanus-like synanamorphs not observed.

Distribution. Currently known from Kunming City and Puer City in Yunnan Province, China.

Other Material Examined. China, Yunnan Province, Puer City (22.58° N, 99.97° E, alt. 1287 m), on a spider attached to the underside of a leaf, August 2025, Hui Chen (GMB 3143; living culture: not available).

Notes. In the phylogeny, G. yunnanensis clusters with G. attenboroughii and G. flava, forming a highly supported sister clade with G. attenboroughii (IQ-TREE-BS/RAxML-BS/BI-PP = 99%/90%/0.92). Morphologically, G. yunnanensis resembles G. attenboroughii and G. flava in producing multiple synnemata and aspergillate, distinctly roughened conidiophores. However, G. yunnanensis differs from G. attenboroughii in several stable morphological features. The conidia of G. yunnanensis are distinctly longer and narrower, whereas those of G. attenboroughii are ellipsoidal to fusoid (6–9 × 2.2–3.1 μm vs. 4–6 × 1.5–2 μm). In addition, the conidial heads of G. yunnanensis are densely arranged, while those of G. attenboroughii are relatively loose. These consistent morphological distinctions, combined with its stable phylogenetic position, support the recognition of G. yunnanensis as an independent species.

4. Discussion

In China, research on spider-pathogenic fungi has a relatively long history, yet the diversity of Gibellula has only recently gained fuller appreciation. By the 1980s, only a single species—initially reported as G. pulchra [51]—was known. This record was later revised to G. leiopus, a species characterized by extremely short conidiophores that confer a compact appearance [20]. Subsequent studies in the 1990s described several new taxa from Taiwan and Anhui Province, and over the past decade, systematic work has significantly expanded the known diversity [40,45,50]. To date, a total of 14 species and/or varieties of Gibellula have been reported from China, including G. clavispora, G. clavulifera, G. clavulifera var. major, G. curvispora, G. dabieshanensis, G. dimorpha, G. flava, G. leiopus, G. longispora, G. penicillioides, G. pulchra, G. shennongjiaensis, G. unica, and G. liaoningensis [14,17,19,45,50,52]. Among these, G. pulchra and G. leiopus are commonly encountered in southern China. The discovery of the five new species in this study, particularly those from Jilin and Liaoning Provinces, therefore significantly contributes new distribution records for Gibellula in Northeast China and underscores the ongoing potential for taxonomic discovery even in previously studied geographic contexts.

Host specificity offers critical insights for evaluating both the virulence of pathogens and their potential application as biological control agents [40]. Concurrently, increasing attention is being directed towards the secondary metabolites produced by Gibellula species. For instance, the novel antimicrobial compound EPF083CE was isolated from G. pulchra EPF083 [53]; pigmentosins A and B were obtained from the spider-associated fungus G. pigmentosinum [54]; and gibellamines A and B were characterized from G. gamsii [43]. Notably, pigmentosin B and gibellamines appear to be unique to G. pigmentosinum and G. gamsii, respectively, suggesting their potential utility as chemotaxonomic markers [40].

Members of Gibellula are notoriously fastidious and often challenging to establish in pure culture. In this study, however, we achieved successful isolation of the majority of the newly described taxa, with four out of the five species grown on potato dextrose agar (PDA) from conidial sources, despite their generally slow growth rates. This cultivation success represents a significant step forward, paving the way for utilizing these isolates in the discovery of novel bioactive metabolites and for identifying chemical markers valuable in taxonomic studies.