Abstract
Cudonia and Pleurocordyceps comprise a relatively limited number of species worldwide. In particular, Pleurocordyceps represents an ecologically important group of entomopathogenic fungi with considerable potential for medicinal applications. In this study, two novel species, Cudonia aurantiaca and Pleurocordyceps tianmushanensis, were collected from the National Nature Reserve of Mount Tianmu, Zhejiang Province, China. Detailed morphological descriptions and illustrations are provided, and genus-level phylogenetic relationships are inferred based on a combined multi-locus sequence dataset comprising the internal transcribed spacer (ITS), small subunit ribosomal RNA (SSU), large subunit ribosomal RNA (LSU), translation elongation factor 1-alpha (tef-1α), RNA polymerase II largest subunit (rpb1), and RNA polymerase II second largest subunit (rpb2) gene regions. This study contributes to a better understanding of the diversity and taxonomy of Cudonia and Pleurocordyceps.
Keywords: entomopathogenic fungi, morphology, multigene fragment, phylogeny, taxonomy
1. Introduction
Cudonia belongs to Cudoniaceae, Rhytismatales, Leotiomycetes, and was established by Fries in 1849, with the type species C. circinans [1]. Initially, Cudonia was placed in Geoglossaceae based on its rounded to clavate ascomata and cylindrical stipe [2,3,4]. The asci are clavate, J-, 8-spored. Paraphyses are filiform, curved to circinate, or occasionally straight distally. Ascospores are filiform to acicular, becoming one- to multiseptate at maturity [3,4]. However, because morphological differences among species are subtle, the systematic position of the genus remained controversial in early taxonomic treatments [2,3,4,5,6,7,8,9,10]. With advances in molecular phylogenetics, the taxonomic placement of Cudonia has received increasing research attention [11,12,13,14,15]. In 2001, Kirk et al. [16] established the family Cudoniaceae (Helotiales, Ascomycotina), with Cudonia as the type genus. Furthermore, molecular phylogenetics has confirmed that Cudoniaceae should be placed in Rhytismatales [3,17].
Species of Cudonia are widely distributed in temperate regions, with a few species extending into subtropical zones, and they typically occur in coniferous forests or on humus layers associated with mosses [3]. To date, 19 species of Cudonia have been recorded in Index Fungorum (https://indexfungorum.org/Names/Names.asp, accessed 20 March 2026). Notably, several Cudonia species lack molecular data, which hinders comprehensive taxonomic and phylogenetic studies of the genus [15].
Pleurocordyceps is placed in Polycephalomycetaceae, Hypocreales, Sordariomycetes, and was established by Wang et al. [18] with the type species P. sinensis. Pleurocordyceps is the most species-rich genus within the family Polycephalomycetaceae, with 25 species currently recorded in Index Fungorum (https://indexfungorum.org/Names/Names.asp, accessed 20 March 2026) [18,19]. In China, 15 species of Pleurocordyceps have been reported [19,20]. The genus is distinguished from closely related taxa by the presence of laterally fertile, pulvinate stromata near the apex of the sexual morph, as well as by the occurrence of two distinct types of phialides and conidia in the asexual morph [18,19,20,21]. Species of Pleurocordyceps have been reported from a wide range of insect hosts, including members of Coleoptera, Hymenoptera, Hemiptera, Lepidoptera, Orthoptera, and Homoptera [11,22,23]. In addition to parasitizing insects, most species in the genus are also parasites of fungi [20,21,24]. Species of Pleurocordyceps exhibit considerable potential for the production of diverse secondary metabolites [21,24].
Over the past decades, several novel species of Cudonia and Pleurocordyceps have been described [4,15,19,20,21]. Nevertheless, a portion of the species diversity within both genera remains undocumented [4,25]. In this study, we describe two novel species, Cudonia aurantiaca and Pleurocordyceps tianmushanensis, collected from the National Nature Reserve of Mount Tianmu, Zhejiang Province, China. Detailed morphological descriptions and phylogenetic analyses are provided to support their taxonomic placement.
2. Materials and Methods
2.1. Sample Collection, Isolation and Morphological Studies
Fresh specimens were collected from the National Nature Reserve of Mount Tianmu, Zhejiang Province, China, during the period 2023–2025. For Pleurocordyceps species, one fresh sample was collected, and more than ten samples of Cudonia species were collected from the same locality. The elevation of the reserve ranges from approximately 300 to 1500 m, with abundant vegetation. Vegetation types include mixed coniferous broadleaved forest (300–1200 m, a.s.l.), mixed evergreen deciduous broadleaved forest (800–1100 m, a.s.l.), and deciduous broadleaved forest (1100–1500 m, a.s.l.). The reserve has an average annual temperature range from 8.8 to 14.8 °C, with annual precipitation between 1390 and 1870 mm [26].
Between June and August 2025, for Pleurocordyceps species, strains were isolated from fresh specimens by aseptically excising small pieces of mycelium from the host and transferring them onto PDA plates using a sterile needle. The cultures were incubated at 25 °C until pure cultures were obtained. The pure culture was deposited in the Herbarium of Hangzhou Normal University (HTC). The strain of Cudonia species was not isolated. All voucher specimens are stored in HTC.
The macroscopic characteristics and microstructure are described for two samples of Cudonia, one sample and two strains of Pleurocordyceps species. Micromorphological observations were performed using a super depth-of-field microscope (KEYENCE VHX-6000, Osaka, Japan) and a compound microscope (Eclipse Ni, Belfast, UK). The colors of the specimens were coded according to Kornerup & Wanscher [27]. For Cudonia species, microscopic structures were obtained directly from free-hand sections of the dried ascomata and observed in Lactophenol Cotton Blue (LPCB). For Pleurocordyceps species, fresh strain samples were observed in LPCB as well. The morphological structures were measured with 20 measurements using ImageJ (v.1.54m, National Institutes of Health, Bethesda, MD, USA). Ranges in the measurements of microcharacters are presented as ranges from the minimum to the maximum values. The photographic plates were processed using Adobe Photoshop CC 2020 (Adobe Systems, San Jose, CA, USA).
2.2. DNA Extraction, PCR Amplification and Sequencing
Total DNA was extracted from ascomata and strains using the Ezup Column Fungal Genomic DNA Extraction Kit (Sangon Co., Ltd., Shanghai, China), following the manufacturer’s instructions. ITS, SSU, LSU, rpb1, rpb2, and tef-1α gene amplifications were performed using the ITS1/ITS4, NS1/NS4, LR0R/LR5, CRPB1A/RPB1Cr, fRPB25F/fRPB2-7Cr, and 983F/2218R primers, respectively [28,29,30,31,32,33,34]. The nuclear gene amplification reactions followed the protocol outlined by Yang et al. [35]. The PCR products were sequenced by Sangon Biotech (Shanghai, China). All newly generated sequences were assembled using SeqMan. Sequence quality was assessed using BioEdit (v.7.7.1) and BLAST searches following the procedures described by Nilsson et al. [36]. The new sequences were uploaded to GenBank, and accession numbers were assigned (Table 1 and Table 2).
Table 1.
Sequence information and GenBank numbers of samples used in Cudonia study.
Notes: Newly generated sequences are indicated in bold. “-” means no data available in GenBank. The taxa marked with a “T” are type specimens.
Table 2.
Sequence information and GenBank numbers of samples used in Pleurocordyceps study.
| Taxon | Strain | ITS | SSU | LSU | rpb1 | rpb2 | tef-1α | Country |
|---|---|---|---|---|---|---|---|---|
| Perennicordyceps cuboidea | NBRC 10383 | JN943319 | JN941735 | JN941406 | JN992469 | AB968563 | AB968602 | Japan |
| Pe. cuboidea | NBRC 101740 | JN943321 | JN941734 | JN941407 | JN992468 | AB968564 | AB968603 | Japan |
| Pleurocordyceps agarica | YHCPA1307 | KP276654 | - | - | KP276666 | KP276670 | KP276662 | China |
| P. aurantiaca | GZU 20-2306 | OQ172069 | - | OQ172041 | - | OQ459789 | - | Thailand |
| P. aurantiaca | MFLU 17-1394 | MG136918 | MG136906 | MG136912 | MG136867 | MG136872 | MG136876 | Thailand |
| P. aurantiaca | MFLU 21-0276 | OQ172072 | - | OQ172042 | - | OQ459788 | Thailand | |
| P. aurantiaca | MFLUCC 17-2113 | MG136916 | MG136904 | MG136910 | MG136866 | MG136870 | MG136875 | Thailand |
| P. clavisynnema T | GZLG 20-102 | OQ968788 | - | OQ968796 | - | - | OQ982009 | China |
| P. clavisynnema | GZCC 22-2042 | OQ968789 | OQ968805 | OQ968797 | OQ981998 | OQ982004 | OQ982008 | China |
| P. formosus | MFLU 18-0162 | MK863250 | MK863043 | MK863050 | - | - | - | China |
| P. heilongtanensis | HKAS:148549 | PV701341 | PV791134 | PV701302 | - | - | - | China |
| P. heilongtanensis | HKAS:148554 | PV701372 | PV702211 | PV701332 | - | - | - | China |
| P. heilongtanensis | HKAS:148574 | PV737571 | PV739068 | - | - | PV740523 | PV740522 | China |
| P. heilongtanensis | KUMCC 3008 | OQ172091 | OQ172111 | OQ172063 | OQ459759 | OQ459805 | OQ459731 | China |
| P. lanceolatus | GACPCC 17-2005 | OQ172077 | - | OQ172047 | OQ459755 | OQ459801 | OQ459727 | China |
| P. lianzhouensis | GIMYY9603 | EU149922 | KF226249 | KF226250 | KF226251 | - | KF226252 | - |
| P. lianzhouensis | HIMGD20918 | EU149921 | KF226245 | KF226246 | KF226247 | - | KF226248 | - |
| P. marginaliradians | MFLU 17-1582 | MG136920 | MG136908 | MG136914 | MG136869 | MG271931 | MG136878 | Thailand |
| P. marginaliradians | MFLUCC 17-2276 | MG136921 | - | MG136915 | - | - | MG136879 | Thailand |
| P. multisynnema | GZCC 22-2041 | OQ968793 | OQ968803 | OQ968801 | OQ981997 | OQ982003 | OQ982010 | - |
| P. multisynnema T | GZLG 23-101 | OQ968792 | OQ968802 | OQ968800 | - | OQ982002 | - | China |
| P. neoagarica | GZCC 22-2043 | OQ968791 | OQ968804 | OQ968794 | OQ981996 | OQ981999 | OQ982007 | China |
| P. neoagarica T | GZLC23-103 | OQ968790 | - | OQ968795 | - | - | - | China |
| P. neoagarica | HKAS:148550Y | PV701361 | PV791136 | PV701323 | - | - | - | China |
| P. nipponica | HKAS 100058 | MG029523 | MG725823 | MG725825 | - | - | - | China |
| P. nipponi ca | NHJ 4268 | KF049657 | KF049621 | KF049639 | MF416676 | KF049676 | MF416517 | - |
| P. nutansis | GACP 19-1906 | OQ172079 | OQ172117 | OQ172049 | OQ459763 | OQ459809 | OQ459737 | China |
| P. nutansis T | MFLU 21-0275 | OQ172073 | OQ172119 | OQ172048 | OQ459765 | OQ459811 | OQ459739 | China |
| P. onorei | BRA CR23902 | KU898841 | - | - | - | - | - | Ecuador |
| P. onorei | BRA CR23904 | KU898843 | - | - | - | - | - | Ecuador |
| P. phaothaiensis | BCC84557 | MF959733 | - | MF959737 | MF959745 | - | MF959742 | Thailand |
| P . puerensis | GACPY117 | PP627512 | PP707759 | PP616709 | PP654216 | - | PP654214 | China |
| P. ramosopulvinat a | EFCC 5566 | KF049658 | - | KF049627 | KF049645 | - | KF049682 | - |
| P. ramosus | NBRC 101760 | MN586827 | MN586818 | MN586836 | MN598042 | MN598060 | MN598051 | - |
| P. sinensis | MFLU 21-0270 | - | - | - | OQ459751 | OQ459796 | - | China |
| P. sinensis | MFLU 21-0271 | - | - | - | OQ459752 | OQ459797 | - | China |
| P. sanduensis | GZCC 22-2044 | OQ968787 | OQ968806 | OQ968799 | OQ981995 | OQ982001 | OQ982006 | China |
| P. sanduensis T | GZLG 23-104 | OQ968786 | - | OQ968798 | - | OQ982000 | OQ982005 | China |
| P. tianmushanensis T | SYYCC001 | PZ070746 | PZ070751 | PZ134459 | PZ124379 | PZ095765 | PZ124381 | China |
| P. tianmushanensis | SYYCC002 | PZ070747 | PZ070752 | PZ134460 | PZ124380 | PZ095766 | PZ124382 | China |
| P. tomentosus | BL4 | KF049666 | KF049623 | KF049641 | KF049656 | KF049678 | KF049697 | - |
| P. vitellina | KUMCC 3005 | OQ172088 | - | OQ172060 | OQ459756 | OQ459802 | OQ459728 | China |
| P. vitellina | KUMCC 3006 | OQ172089 | - | OQ172061 | OQ459757 | OQ459803 | OQ459729 | China |
| Pleurocordyceps sp. | GZU 19-1907 | OQ172087 | - | - | OQ459764 | - | China | |
| Pleurocordyceps sp. | GZU 19-3019 | OQ172086 | - | - | OQ459766 | OQ459812 | - | China |
| Pleurocordyceps sp. | YFCC 07239279 | - | - | - | PP581807 | PP581824 | PP254877 | China |
| Pleurocordyceps sp. | YFCC 07239280 | - | - | - | PP581808 | PP581825 | PP254878 | China |
Notes: Newly generated sequences are indicated in bold. “-” means no data available in GenBank. The taxa marked with a “T” are type specimens.
2.3. Phylogenetic Analyses
Reference taxa for phylogenetic analyses were selected based on BLAST search results (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed 20 March 2026) and previously published datasets [11,15,19]. Individual loci were aligned using MAFFT v.7 (https://mafft.cbrc.jp/alignment/server, accessed 20 March 2026) and manually adjusted and trimmed in BioEdit [37]. Phylogenetic relationships were inferred using maximum likelihood (ML) and Bayesian inference (BI) analyses. ML analysis was conducted using RAxML-HPC2 v.8.2.12 on the CIPRES Science Gateway web server (http://www.phylo.org/portal2, accessed 20 March 2026), employing 1000 rapid bootstrap replicates and the GTRGAMMA+I model. For the ML analysis, bootstrap support values of 75% or higher were noted above the nodes in the phylogenetic tree. BI phylogenetic analyses were determined by Markov Chain Monte Carlo (MCMC) sampling using MrBayes on ACCESS via the CIPRES science gateway (http://www.phylo.org, accessed 20 March 2026). BI analyses were performed with six independent MCMC runs in our operation, and trees were sampled every 100 generations. The analyses were stopped after 5,000,000 generations when the average standard deviation of split frequencies was below 0.01. The first 25% of the resulting trees were discarded as burn-in and posterior probabilities (PP) were calculated from the remaining sampled trees. Outgroup taxa were selected following previous related studies [15,19]. The ML and Bayesian trees were visualized with FigTree v.1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/, accessed 20 March 2026).
3. Results
3.1. Phylogenetics
As the topologies of ML and BI trees were similar, and only the ML tree is presented here (Figure 1 and Figure 2). Phylogenetic analyses of Cudonia species were conducted based on a combined dataset of ITS, LSU, rpb2, and tef-1α sequences from 55 samples representing 7 Cudonia species. The 55 samples of Cudonia, which included two newly generated sequences of Cudonia (T587 and T5872), formed a distinct clade with strong statistical support (BS = 97%, PP = 1; Figure 1). Samples of C. aurantiaca formed a distinct clade with strong statistical support (BS = 100%, PP = 1). Cudonia aurantiaca was recovered as sister to Cudonia sp. (H680) with low bootstrap support (BS = 78%, PP = 0.94). In addition, C. aurantiaca clustered with C. lutea in a well-supported lineage (BS = 81%, PP = 0.98).
Figure 1.
ML phylogenetic tree of Cudonia combining ITS, LSU, rpb2 and tef-1α sequence data. The tree is rooted to Spathulariopsis velutipes (H338) and Spathularia flavida (C308). ML bootstrap values ≥ 75% and PP from Bayesian Inference ≥ 0.95 are given above branches. Cudonia aurantiaca is indicated in pink. The taxa marked with a “T” are type specimens. Cudonia is highlighted in light blue, while the outgroup is marked with a light green background.
Figure 2.
Phylogram of the Pleurocordyceps clade generated by Maximum Likelihood (ML) analysis of ITS, LSU, SSU, tef-1α, rpb1, and rpb2 sequence data, showing the position of P. tianmushanensis (indicated in pink). The tree is rooted to Perennicordyceps cuboidea (NBRC 101740) and Pe. cuboidea (NBRC 10383). ML bootstrap values ≥ 75% and PP from Bayesian Inference ≥ 0.95 are given above branches. The taxa marked with a “T” are type specimens. Pleurocordyceps is highlighted in light yellow, while the outgroup is marked with a light green background.
Phylogenetic trees of Pleurocordyceps were constructed using a combined six-gene dataset, including ITS, SSU, LSU, rpb1, rpb2 and tef-1α sequence data from 45 samples representing 22 Pleurocordyceps species. In the phylogenetic analysis of Pleurocordyceps, the samples (SYYCC001 and SYYCC002) formed a distinct and well-supported lineage (BS = 99%, PP = 1; Figure 2). Pleurocordyceps tianmushanensis was recovered as sister to P. sanduensis and P. puerensis with strong statistical support (BS = 86%, PP = 0.96). In addition, Pleurocordyceps tianmushanensis, P. sanduensis, P. puerensis, and P. clavisynnema formed a distinct clade with strong statistical support (BS = 98%, PP = 1; Figure 2).
3.2. Taxonomy
Cudonia aurantiaca Y.Y. Shen & Y.B. Song, sp. nov. Figure 3 and Figure 4.
Figure 3.
(a) Habitat of Cudonia aurantiaca. (b) Surface of sipe. (c) Asci and ascospores. (d) Paraphyses. (e) Conidia and one ascospore producing conidia.
Figure 4.
(a) Drawing of ascomata. (b) Asci and ascospores. (c) Ascospores. (d) Conidia and three ascospores producing conidia. (e) Paraphyses. (f) Longitudinal section of the stipe on its surface.
Index Fungorum number: IF905160; Facesoffungi number: FoF 19637.
Etymology: The specific epithet “aurantiaca” refers to the orange color of the ascomata.
Holotype: HTC (collecting numbers T587), China, Zhejiang Province, Hangzhou, National Nature Reserve of Mount Tianmu at 1176 m a.s.l., 30.34 N, 119.43 E, 25 September 2023, Yu-Yu Shen. Scattered in litter under mixed conifer-broadleaf forest. GenBank accessions: PZ070749 (ITS), PZ125084 (LSU), PZ071831 (SSU), PZ124377 (rpb2), and PZ124383 (tef-1α).
Diagnosis: Cudonia aurantiaca is somewhat similar to C. mongolica. However, C. aurantiaca has a darker color in the saddle-shaped pileus and a darker color in the stipes, covered with obvious and small white spines on the surface of the stipe.
Description: Ascomata gregarious or cespitose, saddle-shaped, stipitate, the edge rolling toward the stipe, 15.3–57.2 mm high. Ascigerous portion capitate, semiorbicular, 11.2–57.2 mm diam. When young, the surface of hymenium light orange to golden yellow (5A4-5A6), greyish orange (5B5-5B6) to brownish orange (5C5-5C6) when old, smooth. Receptaculum pale orange to light orange (5A2-5A5). Stipe 11.1–17.4 × 4.7–74.4 mm, subcylindrical, slightly swollen base, greyish orange (5B4-5B5) when young, then pale orange to light orange (5A2-5A5) with growing, smooth or apical part with longitudinal striations, covered with obvious small white spines.
Hymenium about 200 μm thick, consisting of paraphyses and asci at different stages of development. Asci clavate, 88.1–187.7 × 9.5–17.5 μm (x = 101.6 × 12.8 μm, n = 20), clavate to narrowly clavate, attenuated towards the base, 8-spored, apical portion narrowly round, J-, croziers present. Ascospores 39.5–89.6 × 1.4–3.2 μm (x = 69.7 × 2.3 μm, n = 20), clavate-filiform to acicular, colorless and hyaline, thin-walled, round at apex, acuminate at base, multiguttulate, non-septate, the wall with a gelatinous layer 1–2 μm thick. Ascoconidia 1.1–2.4 × 1.1–2.1 μm (x = 1.7 × 1.5 μm, n = 20), subglobose to obovoid, smooth, thin-walled, 1-celled, 1-guttulate, colorless and hyaline, sometimes nearly filling the asci. Paraphyses filiform, often branched and anastomosing below, strongly curved to circinate above, lower portion about 2.5 μm in diam, apical portion about 1.5 μm in diam.
Habitat and distribution: Scattered in litter. The fungus is currently known only from its type locality.
Additional material examined: HTC (collecting numbers T5872, paratype), China, Zhejiang Province, Hangzhou, the National Nature Reserve of Mount Tianmu at 1176 m a.s.l., 30.34 N, 119.43 E, 25 September 2023, Yu-Yu Shen. GenBank accessions: PZ070750 (ITS), PZ145085 (LSU), PZ071832 (SSU), PZ124378 (rpb2), and PZ124384 (tef-1α).
Notes: Cudonia aurantiaca is characterized by saddle-shaped ascomata with cylindrical stipes; asci are clavate, J-, 8-spored, ascospores are filiform to acicular, hyaline, one- to several-septate at maturity, and surrounded by a gelatinous sheath. These characteristics are consistent with the diagnostic features of the genus Cudonia [3,4,15]. Cudonia aurantiaca and C. circinans (type species of Cudonia) can be distinguished by ascocarps and stipe characteristics. Ascocarps of C. circinans are cream to dark brown, with striate to ridged and drab to dark brown stipe [5]. Cudonia aurantiaca has saddle-shaped and orange pileus, and greyish orange stipes, especially covered with obvious and small white spines on the surface of the stipe.
According to our phylogenetic analyses, samples of C. aurantiaca are grouped into a single clade with maximum statistical support (BS = 100%, PP = 1, Figure 1) based on ITS, LSU, rpb2, and tef-1α sequence data. Cudonia aurantiaca clustered with C. lutea in a well-supported lineage (BS = 81%, PP = 0.98). Base pair differences with gaps between C. aurantiaca and C. lutea (wz 139) are 7/508 (1.4%) in ITS, 10/799 (1.3%) in LSU. Morphologically, C. lutea and C. aurantiaca are similar, but C. lutea differs in having longer and narrower ascospores (75–80 × 1.5 μm) compared with those of C. aurantiaca (69.7 × 2.3 μm) [38]. Although C. aurantiaca is phylogenetically distant from C. mongolica, the two species share several morphological similarities. Compared with C. mongolica, C. aurantiaca is distinguished by its saddle-shaped ascigerous portion. In addition, C. mongolica is characterized by the presence of small crystals on the surface of the stipe, but C. aurantiaca is covered with obvious and small white spines [15]. Hence, this study introduces C. aurantiaca as a novel species based on morphological and phylogenetic analyses.
Pleurocordyceps tianmushanensis Y.Y. Shen & Y.B. Song, sp. nov. Figure 5.
Figure 5.
Pleurocordyceps tianmushanensis (SYYCC001, Holotype). (a) Overview of P. tianmushanensis. (b) Synnemata on the host. (c) Host of P. tianmushanensis. (d) Upper and back sides of the colony on PDA medium. (e,f) Conidial masses on the culture. (g) Conidiophores. (h) α-phialides. (i) β-phialides. (j) α-conidia. (k) β-conidia.
Index Fungorum number: IF905161; Facesoffungi number: FoF 19638.
Etymology: The specific epithet “tianmushanensis” refers to the locality where the species was collected, Mount Tianmu, Zhejiang Province, China.
Holotype: HTC (collecting numbers: SYYCC001), China, Zhejiang Province, Hangzhou, National Nature Reserve of Mount Tianmu at 300 m a.s.l., 30.32 N, 119.45 E. Parasitic on larvae of Scarabaeidae species (Coleoptera), grown in soil under mixed conifer-broadleaf forest, collected on 18 June 2025, Zi-Wen Zhang. GenBank accessions: PZ070746 (ITS), PZ070751 (LSU), PZ134459 (SSU), PZ124379 (rpb1), PZ095765 (rpb2), and PZ124381 (tef-1α).
Description: Sexual morph: Not observed. Host 21.90 × 1.8–4.48 mm, a larva of Scarabaeidae species (Coleoptera), characteristically C-shaped, brown. Stromata arising from the head of the host, branched, cylindrical, light yellow to yellow (5A8), 41.05 × 2.10–4.10 mm, with sterile apical. Stipes 39.05 × 2.10–4.10 mm, clavate, the color gradually becoming lighter towards the apex, light yellow to orange (4A7), with hemispherical, cap-shaped parts (4.76–5.24 × 1.52–1.71 mm). Asexual morph: Hyphomycetous. Culture characteristics: Colonies on PDA fast-growing, obtained from tissue, reaching 5 cm wide in 15 days at 28 °C, white, reverse yellow (5A7) to brown (5C8), presenting multiple radiating ring-like patterns. Synnemata emerging after 20 days, solitary, unbranched, 0.2–0.6 mm long, distributed at the edge, with small or without a fertile head. Conidial masses covered the surface of the colony, pale yellow (4A3) when young, later change to brown color (4C4), slime. Conidiophore 21–42 μm long (x = 31.4 μm, n = 30). Phialides exist in α-phialides and β-phialides. α-phialides 18.6–59.3 × 0.7–2.4 μm (x = 34.6 × 1.4 μm, n = 20), smooth, hyaline, solitary. β-phialides 25.0–84.7 × 0.9–2.9 μm (x = 47.0 × 1.6 μm, n = 20), smooth, hyaline, solitary. α-conidia 0.8–2.1 μm (x = 1.4 μm, n = 30) wide, globose, unicellular, smooth-walled; β-conidia 1.7–3.5 × 0.8–1.6 μm (x = 2.7 × 1.3 μm, n = 30) fusiform, unicellular, hyaline, smooth-walled.
Habitat and distribution: Scattered in soil. The fungus is currently known only from its type locality.
Material examined: China, Zhejiang Province, Hangzhou, the National Nature Reserve of Mount Tianmu. Parasitic on larvae of Scarabaeidae species (Coleoptera), in soil, collected on 18 June 2025, Zi-Weng Zhang, CC001 (HTC strain numbers: SYYCC001). Ex-type living culture, HTC (strain numbers: SYYCC002). GenBank accessions (SYYCC002): PZ070747 (ITS), PZ070752 (LSU), PZ134460 (SSU), PZ124380 (rpb1), PZ095766 (rpb2), and PZ124382 (tef-1α).
Notes: Pleurocordyceps tianmushanensis is distinguished by the presence of two types of phialides and conidia in the asexual morph, a key diagnostic feature shared with other species of Pleurocordyceps [21]. Pleurocordyceps tianmushanensis and P. sinensis (type species of Pleurocordyceps) are differentiated by synnemata. Synnemata of P. sinensis were bigger compared to P. tianmushanensis (50–60 vs. 2–10 mm, respectively), clavate and capitate [18].
According to our phylogenetic analyses, samples of Pleurocordyceps tianmushanensis are sister to P. sanduensis and P. puerensis with high statistical support (BS = 92%, PP = 1, Figure 2). Samples of Pleurocordyceps tianmushanensis, P. sanduensis, P. puerensis and P. clavisynnema formed a distinct clade with strong statistical support (BS = 86%, PP = 0.96; Figure 2). The four species are closed in phylogeny. Base pair differences with gaps between P. tianmushanensis and P. sanduensis are 1/607 (0.2%) in ITS, 0/734 in LSU, 7/1082 (0.6%) in SSU, 7/846 (0.8%) in tef-1α, and 4/918 (0.4%) in rpb2 (Table 3). Between P. tianmushanensis and P. puerensis, the differences are 4/512 (0.8%) in ITS, 0/716 in LSU, 15/798 (1.9%) in SSU, 5/855 (0.6%) in tef-1α, and 1/594 (0.2%) in rpb1 [25]. Base pair differences between P. tianmushanensis and P. clavisynnema are 1/594 (0.2%) in ITS, 0/1231 in SSU, 0/856 in LSU, 2/642 (0.3%) in rpb1, 4/968 (0.4%) in rpb2, and 4/878 (0.5%) in tef-1α. We further compared the base pair differences with gaps among the other three species (P. sanduensis, P. puerensis, and P. clavisynnema), and the results show that the differences among these species are small, especially for the ITS and LSU (Table 3). Specifically, Pleurocordyceps sanduensis differs from P. clavisynnema by 0/518 in ITS, 0/815 in LSU, 8/1130 (0.3%) in SSU, 2/678 (0.3%) in rpb1, 3/1050 (0.3%) in rpb2, and 23/824 (2.8%) in tef-1α [18,19]. Pleurocordyceps sanduensis differs from P. puerensis by 2 bp in ITS, 0 bp in LSU, 1 bp in rpb1, and 6 bp in tef-1α. Between P. clavisynnema and P. puerensis, the differences are 3/552 (0.5%) in ITS, 0/764 in LSU, 0/800 in SSU, 1/676 (0.1%) in rpb1, and 7/795 (0.9%) in tef-1α [19,25]. Pleurocordyceps tianmushanensis was isolated from scarabaeid larvae, while P. sanduensis and P. clavisynnema parasites on Ophiocordyceps neogryllotalpae; Pleurocordyceps puerensis parasites on larvae of Coleoptera. Morphologically, P. tianmushanensis differs from P. sanduensis in larger phialides (α-phialides: 34.56 × 1.41 vs. 14.1 × 1.45 μm; β-phialides: 47.0 × 1.6 vs. 26.2 × 1.35 μm, respectively), and smaller conidia (α-conidia: 1.4 vs. 2.6 μm; β-conidia: 2.7 × 1.3 vs. 4 × 1.8 μm, respectively) [19,25]. Pleurocordyceps tianmushanensis differs from P. puerensis in orange stipe [25]. Due to the lack of sexual morphology of P. tianmushanensis, it is impossible to compare P. tianmushanensis and P. puerensis in morphological characteristics [25]. Hence, this study introduces P. tianmushanensis as a novel species based on morphological and phylogenetic analyses.
Table 3.
Base pair differences in Pleurocordyceps tianmushanensis, P. sanduensis, P. puerensis, and P. clavisynnema with gaps.
| Species Pair | ITS | SSU | LSU | rpb1 | rpb2 | tef-1α |
|---|---|---|---|---|---|---|
| P. tianmushanensis vs. P. sanduensis | 1/607 (0.2%) | 7/1082 (0.6%) | 0/734 | - | 4/918 (0.4%) | 7/846 (0.8%) |
| P. tianmushanensis vs. P. puerensis | 4/512 (0.8%) | 15/798 (1.9%) | 0/716 | 1/594 (0.2%) | - | 5/855 (0.6%) |
| P. tianmushanensis vs. P. clavisynnema | 1/594 (0.2%) | 0/1231 | 0/856 | 2/642 (0.3%) | 4/968 (0.4%) | 4/878 (0.5%) |
| P. sanduensis vs. P. puerensis | 2/607 (0.3%) | 17/701 (0.1%) | 0/821 | 1/672 (0.1%) | - | 6/867 (0.7%) |
| P. sanduensis vs. P. clavisynnema | 0/518 | 8/1130 (0.3%) | 0/815 | 2/678 (0.3%) | 3/1050 (0.3%) | 23/824 (2.8%) |
| P. puerensis vs. P. clavisynnema | 3/552 (0.5%) | 0/800 | 0/764 | 1/676 (0.1%) | - | 7/795 (0.9%) |
4. Discussion
We described two novel species, Cudonia aurantiaca and Pleurocordyceps tianmushanensis, using morphological observations and phylogenetic analyses. Cudonia aurantiaca is characterized by saddle-shaped ascomata with cylindrical stipes; asci are clavate, J-, 8-spored, consistent with the diagnostic features of the genus Cudonia [3,4,15]. Two novel species were collected from the National Nature Reserve of Mount Tianmu, Zhejiang Province, China. Cudonia species typically occur in humus layers and are widely distributed in temperate regions, with a few species in subtropical zones [8]. Consistent with this pattern, Cudonia aurantiaca is scattered in litter under mixed conifer-broadleaf forest in subtropical zones. Pleurocordyceps tianmushanensis grown in soil under mixed conifer-broadleaf forest as well. Pleurocordyceps species has a wide range of hosts, including insects (e.g., species of Coleoptera, Hymenoptera, Hemiptera, Lepidoptera, Orthoptera, and Homoptera) and fungi (e.g., species of Ophiocordyceps) [20]. Consistently, Pleurocordyceps tianmushanensis parasitized on larvae of Coleoptera species. The extensive host ranges and distinctive biogeographic distributions of Pleurocordyceps further reflect their high ecological adaptability. In addition, species of Pleurocordyceps exhibit considerable potential for the production of diverse secondary metabolites. For example, P. nipponicus and P. phaothaiensis have been reported to produce natural compounds with antioxidant, antibacterial, antitumorigenic, anti-inflammatory, and antimicrobial activities [20,21,28]. Despite these findings, substantial knowledge gaps remain regarding the chemistry, industrial potential, and ecological roles of Pleurocordyceps species [15]. Therefore, further investigations are warranted to comprehensively explore the diversity, functions, and potential applications of secondary metabolites within this genus.
Based on integrative taxonomic and molecular phylogenetic evidence, this study adds one novel species each to the genera Cudonia and Pleurocordyceps from China, thereby expanding their known geographical diversity and providing a valuable basis for future studies on the evolution and utilization of these genera.
Acknowledgments
We are grateful to Xingqiang Wang and Ziwen Zhang for collecting samples. We would like to thank Yu Yang for her help in the experimentation and Xin-Qiang Guo for his assistance in specimen storage in HTC.
Abbreviations
The following abbreviations are used in this manuscript. The generic names referenced in this study are abbreviated as follows: C. = Cudonia, P. = Pleurocordyceps, Pe. = Perennicordyceps. In the fungal descriptions, the symbol “x” represents the mean value of the measured characteristic; “n” represents the number of measured objects.
Author Contributions
Methodology, Y.-Y.S. and M.D.; Software, Y.-Y.S., Y.-J.C. and Z.-R.D.; Formal analysis, Y.-Y.S., Y.-J.C. and Z.-R.D.; Investigation, Y.-Y.S. and C.-M.P.; Resources, C.-M.P.; Writing—original draft, Y.-Y.S., Y.-J.C., Z.-R.D., J.-Y.D. and Y.-B.S.; Writing—review & editing, Y.-Y.S., J.-Y.D., Y.-B.S. and M.D.; Visualization, Y.-Y.S., Z.-R.D. and C.-M.P.; Supervision, Y.-B.S.; Project administration, Y.-B.S.; Funding acquisition, Y.-B.S. All authors have read and agreed to the published version of the manuscript.
Data Availability Statement
The data presented in the study are deposited in the Herbarium of Hangzhou Normal University, accession numbers T587, T5872, SYYCC001, and SYYCC002.
Conflicts of Interest
The authors declare no conflict of interest.
Funding Statement
This work was supported by Hangzhou Normal University 2025 Graduate Student Research and Innovation Promotion Project (Doctoral Program: grant no. 2025HSDYJSKY010).
Footnotes
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data presented in the study are deposited in the Herbarium of Hangzhou Normal University, accession numbers T587, T5872, SYYCC001, and SYYCC002.