Fungal-infected insect specimens were collected from understory vegetation, stones, and decomposing matter in mixed forests of Fuzhou City, the Gongyashan National Forest Park, Longqishan National Nature Reserve, and Wuyi Mountain National Nature Reserve in Fujian Province, China (Supplementary Figure S1), between September 2023 and June 2024, focusing on the summer months. Infected specimens were recorded and photographed in the field before collection and transport to the laboratory for further analyses. The specimens were stored in 50-mL sterile conical tubes containing a small amount of silica gel. To obtain purified isolates, stromata or synnemata from host bodies were taken in a sterile workbench, soaked in 75% alcohol for 30 s, and then washed twice with sterile water. After using sterilized filter paper to dry the tissue fragment, sample segments were inoculated onto potato dextrose agar (PDA: fresh potato chips 200 g/L, agar and dextrose 20 g/L, respectively) plates. After allowing for fungal mycelial growth (3–5 days), colony edges were used to inoculate fresh PDA plates until a pure culture was obtained by visual inspection. All characterized specimens have been preserved in the Fungarium (HMAS) at the Institute of Microbiology, Chinese Academy of Sciences. Characterized microbial strains have also been deposited with the China General Microbiological Culture Collection Center (CGMCC).

A portion of the tissue from the pure culture (PDA plates) was picked and mounted in acid cotton blue on a slide. The microscopic morphological characteristics of the strains were observed using a Nikon Ni-U (Tokyo, Japan) compound microscope and a Nikon SMZ74 (Tokyo, Japan) stereomicroscope, and measurements were made using Digimizer image analysis software 6.4.0 (MedCalc Software Ltd., Belgium). Fungal cultures were also incubated on a PDA (25°C) for 2 weeks and subsequently photographed using a Canon EOS 6D Mark II (Tokyo, Japan) camera. Colony growth rates were calculated based on the methods of Liu and Hodge by taking measurements daily (Liu and Hodge, 2005).

The total genomic DNA was extracted from the cultured mycelia using the Omega D3390 Fungal DNA Mini Kit (Guangzhou Feiyang Biological Engineering Co., LTD, China) following the manufacturer’s instructions. Five-gene loci including the nuclear ribosomal small subunit (nrSSU) (Wang et al., 2015), the nuclear ribosomal large subunit (nrLSU) (Vilgalys and Hester, 1990; Rehner and Samuels, 1994), translation elongation factor 1-α (tef1-α) (Bischof et al., 2006; Sung et al., 2007), and RNA polymerase II largest and second largest subunits (rpb1 and rpb2) (Liu et al., 1999; Bischof et al., 2006; Sung et al., 2007) were targeted for PCR amplification and sequencing (primers given in Table 1, Sangon Biotech, Shanghai Co., Ltd). Each PCR amplification reaction consisted of 12.5 μL of 2 × Rapid Taq PCR Master Mix (Vazyme, Nanjing, China), 1 μL of each forward and reverse primer (10 μM), 1 μL of genomic DNA, and 9.5 μL of distilled deionized water (Sangon Bio Co., Ltd., Shanghai, China) to a total volume of 25 μL. PCR reactions were performed in a Bio-Rad thermal cycler (Hercules, CA, United States). The PCR products were purified and sequenced by Tsingke Biotech Co., Ltd. (Fuzhou, China). Sequences generated in this study have been submitted to GenBank (https://www.ncbi.nlm.nih.gov/genbank, under accession numbers as given in Table 2).

Phylogenetic analyses were performed based on the five genetic loci examined (nrSSU, nrLSU, tef1-α, rpb1, and rpb2) and corresponding sequences from GenBank. GenBank accession numbers of comparative taxa used are listed in Table 2. MAFFT v. 7 online website (http://mafft.cbrc.jp/alignment/server/, accessed on 17 September 2024) was used to align all the sequences, and then sequences were manually edited using BioEdit v.7.2.6.1 (He et al., 2022) and MEGA v.7.0 (Kumar et al., 2016) as needed. The aligned loci sequences were concatenated using Phylosuite v1.2.2 (Zhang et al., 2020). Based on five-gene combination datasets, phylogenetic analyses were performed using Bayesian inference (BI) and maximum likelihood (ML). The ML and Bayesian analyses were conducted using IQ-tree v.2.1.3 (Nguyen et al., 2015) and MrBayes v.3.2.2 (Ronquist et al., 2012), respectively. Four simultaneous Markov chains were run for 2,500,000 generations, and trees were sampled with a frequency of every 100th generation. The initial 25% of sampled trees were discarded as burn-in. The clades support value over 0.9 in BI and greater than 70% in ML were considered significantly supported. Phylogenetic trees were viewed using the ITOL online service¹ and FigTree v.1.4.3 and polished using Adobe Illustrator 2023.

Approximately 40 specimens were collected from four distinct locations in Fujian Province: Gongya Mountain National Forest Park, Longqishan National Nature Reserve, Wuyi Mountain National Nature Reserve, and Fujian Agriculture and Forestry University. More than 35 samples were purified into single colonies, and 12 strains showed unique morphological signatures. Genomic DNA was extracted from these isolates, and selected genetic loci were sequenced, as detailed in the Methods section. Of the 12 specimens examined, 3 were identified as novel isolates (with 1 being a first description in China), as further detailed below.

Five genetic loci (nrSSU, nrLSU, tef1-α, rpb1, and rpb2) were used to construct both ML and BI trees for molecular identification supporting new species descriptions in this study. Pleurocordyceps aurantiaca (MFLUCC 17–2,113) and Pleurocordyceps marginaliradians (MFLU 17–1,582) were selected as outgroups in the phylogenetic reconstruction of Clavicipitaceae. The total length of the concatenated sequences was 4,508 bp (nrSSU: 1–1,042 bp; nrLSU: 1043–1932 bp; tef1-α: 1933–2,829 bp; rpb1: 2830–3,518 bp, and rpb2: 3519–4,508 bp), with analyses including members from 172 taxa from 20 genera. The phylogenetic trees derived from both BI and ML analyses exhibited similar topological structures and indicated three new species and one newly recorded species within Clavicipitaceae as being strongly supported (Figure 1). These molecular analyses revealed that (1) the newly described C. gongyashanensis sp. nov. was closely related to C. fenshuilingensis and C. krungchingensis with high support value (ML-BS: 93%, BI-PP: 0.92), and (2) that two specimens were identical and described in this study as Albacillium fuzhouense, within Albacillium with strong support (BP = 100% and PP = 1). Analyses of four specimens that grouped within the Metarhizium clade indicated a monophyletic cluster that could be assigned to M. cicadae (BP = 94% and PP = 0.99). In addition, two specimens are described in this study as a new species, N. wuyishanensis, which is closely related to its syntopic species N. araneicola, forming a collective group (BP: 99%, PP: 1).

Albacillium fuzhouense L. B. Lin and J. Z. Qiu, sp. nov. (Figure 2).

MycoBank No.: MB856115.

Etymology: Named after Fuzhou City, where the specimen was originally found.

Holotype: China, Fujian Province, Fuzhou City, 119°14′32″ E, 26°4′54″ N, alt. 62 m, found on a larva of Coleoptera attached to the leaf litter in a bamboo garden, 19 June 2024, J. Z. Qiu and L. B. Lin (HMAS353206, holotype; ex-holotype living culture CGMCC3.27818).

Sexual morph: Undetermined.

Asexual morph: Larvae (Coleoptera) covered by white and cottony mycelium, producing white and powdery conidia. Colonies grew well on PDA medium at a relatively constant rate, attaining a diameter of 38–39 mm after 14 days of cultivation at 25°C, with white, felty, dense mycelium, an entire margin, and reverse light yellow. Hyphae were hyaline, smooth-walled, septate, 1.1–2.1 μm wide. Phialides directly originated from aerial hyphae, lanceolate, solitary, hyaline, with a cylindrical base tapering to apex, 13.6–81.5 × 0.8–1.3 μm, mean = 36.9 × 1.1 μm. Conidia were fusiform, ellipsoidal and slightly curved, hyaline, smooth, one-celled, arranged either singly or in mucilaginous spherical clusters at the tips of the phialides, 2.4–6.7 × 1.0–1.4 μm, mean = 4.2 × 1.2 μm.

Habitat: Parasitic on larvae of Coleoptera attached to the leaf litter.

Distribution: China, Fujian Province, Fuzhou City, Jianxin Town.

Other materials examined: China, Fujian Province, Fuzhou City, 119°14′32″ E, 26°4′54″ N, alt. 1,084 m, found on a larva of Coleoptera attached to the leaf litter in a bamboo garden, 19 June 2024, J. Z. Qiu and L. B. Lin (HMAS353207, paratype; ex-paratype living culture CGMCC3.27815).

Notes: The five-gene multilocus phylogenetic analysis revealed that the two samples of A. fuzhouense clustered with A. hingganense with strong support (BP: 100%, PP: 1). Then, the nucleotide sequence comparison of rpb2 showed a 2.1% (23/1091) difference between A. fuzhouense and its related species. Morphologically, A. fuzhouense was similar in shape to its close relatives, yet is distinguished by possessing longer phialides (13.6–81.5 × 0.8–1.3 vs. 8.6–60.1 × 0.9–1.5 μm) and larger conidia (2.4–6.7 × 1.0–1.4 vs. 2.0–4.3 × 0.8–1.3 μm) compared to those of A. hingganense. In addition, the colonies on PDA of A. fuzhouense grew faster than A. hingganense, and the textures of the two are different. A. hingganense is velvet, while A. fuzhouense is more of a felty texture. Moreover, there existed a notable divergence in the host organisms and origins between these two species. The newly found species of the present study were parasitic on the larvae of Coleoptera and were isolated from the Fujian subtropical forest, whereas A. hingganense was isolated from forest litters in a cold temperate climatic zone (Table 3).

Conoideocrella gongyashanensis L. B. Lin and J. Z. Qiu, sp. nov. (Figure 3).

MycoBank No.: MB856116.

Etymology: Named after the Gongya Mountain National Forest Park, where the specimen was originally found.

Holotype: China, Fujian Province, Huaan County, Gongyashan National Forest Park. 117°25′26″ E, 24°54′15″ N, alt. 1,084 m, found on a dead spider that attaching to fallen leaves, 7 May 2024, J. Z. Qiu and L. B. Lin (HMAS353158, holotype; ex-holotype living culture CGMCC3.28305).

Sexual morph: Not observed.

Asexual morph: White to pale yellow and cottony mycelium enveloping the host. Colonies grew rapidly on PDA, reaching a diameter of 32–33 mm in 14 days at 25°C, yellow to light orange, with low mycelium density, a rigid, flocculent margin, and reverse yellowish brown. The growth rate of the colony was 23 mm/day. Hyphae were hyaline, smooth, 1.3–2.5 μm wide. The asexual phase was similar to Hirsutella and originated from vegetative hyphae. These featured conidiogenous structures that had a cylindrical base that gradually narrowed down toward a neck-like extension, hyaline, smooth-walled, 12.7–89.9 × 0.4–1.3 μm, mean = 53.5 × 0.9 μm. Conidia were hyaline, smooth-walled, fusiform-curved, single-celled, and clustered at the apex of the conidiogenous structures, 3.5–10.3 × 1.0–2.5 μm, mean = 6.2 × 1.59 μm.

Habitat: This species was parasitic on scale insects, found attached to the underside of fallen leaves.

Distribution: China, Fujian Province, Huaan County, Gongyashan National Forest Park.

Other materials examined: China, Fujian Province, Huaan County, Gongyashan National Forest Park. 117°25′26″ E, 24°54′15″ N, alt. 1,084 m, found on a dead spider of Sternorrhyncha that attached to the back of living leaves in May 2024, J. Z. Qiu and L. B. Lin (HMAS353159, paratype; ex-paratype living culture CGMCC3.28306).

Notes: C. gongyashanensis is the first species of the genus Conoideocrella to be discovered without an apparent sexual morph. The results of the molecular phylogenetic analysis indicate that two isolates of C. gongyashanensis were found which grouped together, forming a distinct branch within the genus. Phylogenetically, C. gongyashanensis was closely related to C. fenshuilingensis. Therefore, we compared the nucleotides of nrLSU, tef1-α, and rpb1 between C. gongyashanensis and its related species. The differences were as follows: 1.4% (11/812, nrLSU), 6.7% (58/866, tef1-α), and 11.4% (72/630, rpb1) when compared to C. fenshuilingensis.

Metarhizium cicadae S. Mongkolsamrit, A. Khonsanit, D. Thanakitpipattana and J. Luangsa-ard, Stud. Mycol. 95: 212 (2020) (Figure 4).

MycoBank No.: MB856117.

Sexual morph: Not known.

Asexual morph: Cicada adult (Hemiptera) covered by densely bright green powder, produces numerous green and powdery conidia. Colonies grew rapidly on PDA and reached a diameter of 74–75 mm in 14 days at 25°C, at first white and then turning green in the center due to sporulation after 7 days. Colonies flat, floccose, with areas producing green powder, entire margin, white border, reverse as the same, with a growth rate of 5.2–5.3 mm/day. Hyphae were hyaline, branched, smooth-walled, septate, and 1.2–2.6 μm wide. Conidiophores were densely arranged, rising from aerial hyphae, with 2–3 phialides per branch. Phialides cylindrical or clavate, 4.2–10.1 × 1.6–3.0 μm, mean = 6.3 × 2.4 μm, swollen at the bottom, and gradually tapering to a thin neck. Conidia often arrayed in chains, smooth-walled, dimorphic; microconidia came first, ellipsoidal or ovoid, 3.6–6.8 × 2.3–3.2 μm, mean = 5.1 × 2.8 μm; macroconidia produced later, mostly cylindrical, 13.8–20.2 × 2.5–3.6 μm, mean = 17.5 × 3.1 μm.

Habitat: Attached to fallen leaves of dicotyledonous plants or on the trunk of trees.

Distribution: thailand (type country) and China.

Other materials examined: China, Fujian Province, Mingxi County, Gaoyang village, 116°53′38″ E, 26°33′43″ N, alt. 524 m, found on a cicada adult (Hemiptera) covered by green powder, 3 September 2023, J. Z. Qiu and L. B. Lin (HMAS353154, HMAS353155, CGMCC3.28301, CGMCC3.28302 living culture). Fujian Province, Jiangle County, Longqishan National Nature Reserve, 117°20′31″ E, 26°31′43″ N, alt. 1,275 m, found on a cicada adult (Hemiptera), 1 July 2024, J. Z. Qiu and L. B. Lin (HMAS353156, HMAS353157, CGMCC3.28303, and CGMCC3.28304 living culture).

Notes: The species M. cicadae was classified by Mongkolsamrit et al. in 2020, in Thailand (Luangsa-ard et al., 2017). The existence of this species has not been reported in China. In this study, four specimens were collected in Fujian Province, and, according to the phylogenetic analysis, all of them are closely related to M. cicadae with high statistical support (94% in ML and 0.99 in BYPP). The morphology of the four strains was generally similar to M. cicadae, including similarities in the host, phialides proximity, and the size of both macroconidia and microconidia (Table 3). However, the collected specimens exhibited bright green colonies on PDA, attaining 74–75 mm in 14 days, while M. cicadae colonies were cream to green and grew only 15 mm after 14 days of cultivation. Overall, however, our data indicated that the four strains collected in this study should be identified as M. cicadae.

Neoaraneomyces wuyishanensis L. B. Lin and J. Z. Qiu, sp. nov. (Figure 5).

MycoBank No.: MB856118.

Etymology: Named after the Wuyi Mountain National Nature Reserve, where the species was originally found.

Holotype: China, Fujian Province, Wuyishan City, Xingcun Town, Wuyi Mountain National Nature Reserve, 117°39′12″ E, 27°36′14″ N, alt. 952 m, found on a spider adsorbed on the wall, 6 September 2023, J. Z. Qiu and L. B. Lin (HMAS353160, holotype; ex-holotype living culture CGMCC3.28307).

Sexual morph: Undetermined.

Asexual morph: The spider was completely enveloped by white to gray cottony mycelium. Colonies grew readily on PDA, showing velvety, low mycelium density, attaining a diameter of 28–30 mm after 14 days at 25°C. The colony was white, sunken slightly in the middle, reverse, light yellow to orange, with a growth rate of 2.0–2.1 mm/day. Hyphae were hyaline, smooth-walled, septate, 1.0–2.4 μm wide. Phialides were lanceolate, hyaline, originating from aerial hyphae, usually solitary, with a slender base, tapering to a thin neck, 6.2–34.3 × 0.9–2.0 μm, mean = 21.4 × 1.2 μm. Conidia were formed either at the apex of the phialides or in spherical clusters atop the phialides, smooth-walled, hyaline, ellipsoidal, fusiform and slightly curved, one-celled, 2.5–4.5 × 1.0–1.4 μm, mean = 3.1 × 1.2 μm.

Habitat: Attached to stone.

Distribution: China, Fujian Province, Wuyishan City, Xingcun Town, Wuyi Mountain National Nature Reserve.

Other materials examined: China, Fujian Province, Wuyishan City, Xingcun Town, Wuyi Mountain National Nature Reserve, 117°39′12″ E, 27°36′14″ N, alt. 952 m, found on a spider, 6 September 2023, J. Z. Qiu and L. B. Lin (HMAS353161, paratype; ex-paratype living culture CGMCC3.28308).

Notes: Multilocus analyses revealed that the two strains of N. wuyishanensis were similar to N. araneicola (DY101711, ex-holotype) with strong support (99% ML and 1 BYPP), including having the same host, parallel colony morphology, and conidia shape. The nucleotide comparison of rpb1 and rpb2 revealed separation from N. wuyishanensis and N. araneicola with differences of 1.67% (12/720, rpb1) and 1.26% (13/1031, rpb2), respectively. However, the arrangement of conidia between the species was quite different: the conidia of N. wuyishanensis sp. nov. were grouped, while those of N. araneicola were arranged in chains. Concurrently, the phialides of N. wuyishanensis sp. nov. are typically solitary, while those of N. araneicola are either individual or occur in small clusters of two to three. Furthermore, N. wuyishanensis has larger phialides (8.9–23.8 × 1.1–1.6 vs. 10.2–34.3 × 0.9–2.0 μm) and conidia (2.9–4.4 × 1.3–2.0 vs. 2.5–4.5 × 1.0–1.4 μm) compared to N. araneicola. In addition, after 14 days of cultivation on PDA, the mycelium of N. wuyishanensis sp. nov. appeared relatively fluffy, whereas that of its sibling species was slightly more compact. Moreover, this new species produced a pigment that ranged from yellow to orange (Table 3).