Polycephalomycetaceae, a new family of clavicipitoid fungi segregates from Ophiocordycipitaceae

Clavicipitoid fungi comprise three families, namely Clavicipitaceae, Cordycipitaceae, and Ophiocordycipitaceae. They are found worldwide and are specialized pathogens of invertebrate, plant and fungal hosts. Over the last decade, morphology- and phylogeny-based studies on clavicipitoid fungi have increased. The latter have revealed that Polycephalomyces, Perennicordyceps and Pleurocordyceps consistently cluster together. These genera are currently considered as members of Ophiocordycipitaceae. Nonetheless, information with regard to their diversity and ecology remains sparse. To fill this gap, we collected 29 fresh specimens from insect and fungal substrates from tropical and subtropical evergreen forests in Thailand and southwestern China. We performed detailed morphological analyses and constructed photoplates for all isolated fungi. We used extensive taxon sampling and a dataset comprising internal transcribed spacer gene region (ITS), small subunit ribosomal RNA gene region (SSU), large subunit rRNA gene region (LSU), translation elongation factor 1-alpha gene region (TEF-1α), RNA polymerase II largest subunit gene region (RPB1) and RNA polymerase II second largest subunit (RPB2) to infer order-, family and genus-level phylogenetic trees. Based on these biphasic analyses, we segregate Polycephalomyces, Perennicordyceps, and Pleurocordyceps from Ophiocordycipitaceae and introduce the new family Polycephalomycetaceae to accomodate these three genera. The majority of species in this family have a vast range of insect and fungal hosts. The sexual morph of Polycephalomycetaceae has stromatic ascomata, long stipes, thick peridium, and cylindrical secondary spores. The asexual morph is characterized by colonies on the host surface or synnemata with stipes on the host, one or two types of phialides, and cylindrical to fusiform conidia. We expand the number of taxa in the new family by introducing seven new species (Polycephalomyces albiramus, Perennicordyceps lutea, Pleurocordyceps parvicapitata, Pleurocordyceps lanceolatus, Pleurocordyceps nutansis, Pleurocordyceps heilongtanensis, Pleurocordyceps vitellina), nine new hosts, and one new combination (Perennicordyceps elaphomyceticola). The results herein hint at a high level of diversity for Polycephalomycetaceae. Future investigations focusing on obtaining additional collections and specimens from different geographical areas would help to reveal not only the extent of the group’s diversity, but also resolve its deeper phylogenetic placement.


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The type strain from the type species of Polycephalomyces have been changed following evaluation of morphological and molecular data (Kobayasi 1941;Kepler et al. 2013;Wang et al. 2021). Specifically, after updating the molecular data of the type species, Polycephalomyces formosus, and carefully verifying the strains, the epitype (CGMCC 5.2206), lectotype (TNS-F-197965), and neotype (K(M) 187597) were designated and amended (Wang et al. 2021). The morphological analysis using the strain ARSEF 1424 confirmed the type species was considered an erroneous identification, as it produced different conidia from the epitype of Polycephalomyces formosus (Wang et al. 2021). Furthermore, in the updated phylogenetic analysis, ARSEF 1424 placed distantly from the Polycephalomyces clade.
In phylogenetic analyses, Polycephalomyces, Perennicordyceps and Pleurocordyceps consistently formed a distinctive clade, which had maximum statistical support (Wang et al. 2021). The Polycephalomyces clade has claded as sister to either Ophiocordycipitaceae or Clavicipitaceae in the clavicipitoid tree (Wang et al. 2021). Hence, its phylogenetic position is unclear. Some authors have suggested that the three genera might represent a family-level taxon (Kepler et al. 2013;Quandt et al. 2014;Wang et al. 2021). Investigating this clade would provide insights about the evolution, diversity and ecology of clavicipitoid fungi. In recent years, several species have been introduced in Polycephalomyces, Perennicordyceps, and Pleurocordyceps (Kepler et al. 2013;Quandt et al. 2014;Wang et al. 2014Wang et al. , 2015aXiao et al. 2018;Yang et al. 2020). Nonetheless, questions remain with regards to their biodiversity, ecology, and phylogenetic placement.
To fill in some of these gaps, we carried out collections in tropical and subtropical areas in Thailand and China. We provide detailed morphological descriptions along with relevant ecological information at the level of the host and broader habitat for nine species. We carried out extensive phylogenetic analyses using a six genetic marker (LSU, ITS, SSU, TEF-1α, RPB1, RPB2) dataset with a broad and dense sampling of clavicipitoid fungi. The aims of this study are to: (1) infer phylogeny of Ophiocordycipitaceae; (2) investigate the taxonomic rank of the clade including Polycephalomyces, Perennicordyceps, and Pleurocordyceps; (3) illustrate the type species of Polycephalomyces, Perennicordyceps, and Pleurocordyceps; (4) expand the diversity of polycephalomyces-like species.

Specimens and isolates
Twenty-nine fresh specimens were collected along with their substrate (i.e. insects or fungi) from Thailand (n = 2) and southwestern China (n = 27). Collections took place in tropical and subtropical forests, both of which consisted of a variety of evergreen trees. At the time of collection, the temperature was around 15-28 °C, and humidity was > 75%. Fruiting bodies were examined from free-hand sections using a stereomicroscope. A Nikon ECLIPSE 80i and 90i compound microscope fitted with a Canon 550D, 600D, and 750D digital camera was used to examine the micro-morphological characters, including ascomata, perithecia, wall texture, asci, ascospores, secondary spores, conidiophores, phialides (conidiogenous cells), and conidia. Water-mounted slides were prepared for microscopic study. These slides were photographed with a compound microscope using Nomarski microscopy and a Zeiss Axio Imager. Strains were isolated from fresh tissues or single ascospore according to the methods described in Senanayake et al. (2020). Cultures were incubated at 25 °C for 4-10 weeks on potato extract agar (PDA) in ambient daylight to promote sporulation. The Tarosoft (R) v.0.9.7 Image Frame Work was used to measure the size of morphological structures. Photographic plates were edited using the Adobe Photoshop CS6 Extended version 13.0.1 software (Adobe Systems, USA). Averages, standard deviations and analysis of variance (ANOVA) were calculated in Microsoft Excel (Microsoft Excel® version 365, Microsoft Co., Ltd., Redmond, WA., USA). Permanent slides were prepared after checking the morphology according to previously published protocols (Nag Raj 1993;Phookamsak et al. 2014). Dry specimens were deposited in the MFLU (Mae Fah Luang University, Thailand), HKAS (Cryptogamic Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences, China) and GACP (Herbarium of Guizhou University, China) herbarium with silica gel in the particle box.

DNA extraction, PCR amplification, and sequencing
DNA was extracted from fresh specimens and cultures using E.Z.N.A.TM Fungal DNA MiniKit (Omega Biotech, CA, USA) according to the manufacturer's protocols. Moreover, TaKaRakit (TaKaRa Biotechnology, Dalian, China) was used to extract DNA directly from specimens less than 5 mm. Polymerase chain reaction (PCR) was used to amplify six genetic markers using the following primer pairs: ITS4/ITS5 for internal transcribed spacer gene region 1 3 (ITS = ITS1-5.8S-ITS2), NS1/NS4 for partial small subunit ribosomal RNA gene region (SSU), LR0R/LR5 for partial large subunit rRNA gene region (LSU) (Vilgalys and Hester 1990;White et al. 1990), 983F/2218R for partial translation elongation factor 1-alpha gene region (TEF-1α) (Sung et al. 2007b), CRPB1A/RPB1Cr for partial RNA polymerase II largest subunit gene region (RPB1) (Castlebury et al. 2004), and RPB2-5F/RPB2-7R for partial RNA polymerase II second largest subunit (RPB2) (Liu et al. 1999). Amplification reactions were performed in a 50-μl reaction volume as follows: 1 μl of DNA template, 2 μl of each forward and reverse primers (10 pM stock concentration), 25 μl of 2 × Taq PCR SuperMix (TIANGEN BIOTECH Co., Chaoyang District, Beijing, PR China), and 19 μl of sterilized distilled water. The amplification conditions of ITS, LSU, and SSU were as follows: initial denaturation at 94 °C for 3 min, followed by 30 cycles of denaturation at 94 °C for 30-50 s, annealing at 55 °C for 1 min, elongation at 72 °C for 90 s, and final extension at 72 °C for 5-10 min (Liu et al. 2012). The amplification conditions of TEF-1α were as follows: initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 1 min, annealing at 58 °C for 2 min, elongation at 72 °C for 90 s, and final extension at 72 °C for 10 min. Finally, the amplification conditions of RPB1 and RPB2 were the same as those of TEF-1α except for the annealing step, which was carried out at 54 °C for 1 min and 52 °C for 2 min, respectively. The PCR products were purified using DiaSpin PCR Product Purification Kit and sequenced by Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China.

Phylogenetic analysis
DNASTAR Lasergene SeqMan Pro v. 7.1.0 (44.1) was used to edit ambiguous bases at both ends of the raw forward and reverse reads and to assemble them. The newly obtained sequences were used as queries to perform BLAST searches against the nr database to check for contamination, compare species and assemble datasets. MAFFT v.7 was used to align the individual datasets (Katoh et al. 2019, http:// mafft. cbrc. jp/ align ment/ server/). BioEdit (Hall 2011) was used to visually inspect and edit the alignments manually, where necessary. Alignments were trimmed using Trimal (Capella-Gutiérrez et al. 2009).
Six genetic markers, including SSU, ITS, LSU, TEF-1α, RPB1, and RPB2, were used for phylogenetic inferences (Supplementary Tables 1-3). Two trees were inferred for Hypocreales (Table 1) using 213 taxa spanning the diversity of each family: one tree included the ITS region and the other did not. A tree was also inferred for Clavicipitaceae, Ophiocordycipitaceae, Cordycipitaceae, Polycephalomycetaceae, and Calcariosporiaceae (Table 2) using 155 taxa containing the type species of the families. The phylogeny of the new family, Polycephalomycetaceae, was inferred using 95 taxa (Table 3). Three separate trees containing members of Polycephalomyces (Table 3), Perennicordyceps (Table 3). And Pleurocordyceps (Table 3) comprising 17, 30, and 78 taxa, respectively, were also constructed.
Maximum likelihood trees were inferred using IQ-Tree v. 2 (Minh et al. 2020) and models were selected according to Bayesian information criterion (BIC). Partitioned analyses according to genetic marker were carried out for the combined datasets. Branch support was estimated from 1000 ultrafast bootstrap replicates. ModelTest as implemented in MrMTgui (Nuin 2007) was used to determine the best-fit evolution model for Bayesian inference analyses using the Akaike Information Criterion (AIC). MrBayes v.3.1.2 (Ronquist and Huelsenbeck 2003) was utilized to evaluate posterior probabilities (PP) using Markov Chain Monte Carlo sampling (MCMC). The number of generations was determined separately for each dataset and is noted in the individual tree legends. The first 25% of the trees were discarded, as they represented the burn-in phase of the analyses, while the remaining were used for calculating PP in the majority rule consensus tree. For all Bayesian inference trees, convergence was declared when the average standard deviation reached 0.01. The trees were viewed in FigTree v1.4.0 program (Rambaut 2012). The approximately unbiased (AU) test implemented in CONSEL was used to test placement of the newly erected family (Shimodaira and Hasegawa 2001). Topologies with AU test p values less than 0.05 were considered rejected.

Order-level backbone tree
The order level backbone tree ( Fig. 1) was inferred using 213 taxa from Hypocreales, Glomerellales, Chaetosphaeriales, Cordanales, Diaporthales, Conioscyphales and Falcocladiales. Falcocladiales sequences were selected as the outgroup. The final trimmed six genetic marker concatenated dataset contained 4953 bp, including 830 bp for LSU, 519 bp for ITS, 1011 bp for SSU, 901 bp for TEF-1α, 678 bp for RPB1, and 1014 bp for RPB2. The matrix had 3563 distinct patterns, 2542 parsimony-informative, 489 singleton sites, and 1922 constant sites. The likelihood of the best-scoring IQ tree was − 134,632.478. The respective partition best-fit models were TIM + F + I + G4 for LSU, TIM2 + F + I + G4 for ITS, K2P + G4 for SSU, and GTR + F + I + G4 for TEF-1α, RPB1, and RPB2. The GTR model was deemed as the most suitable for Bayesian inference analysis. Six

Five-family tree
Based on the backbone tree in this and other studies (Kepler et al. 2013;Quandt et al. 2014;Ban et al. 2015;Wang et al. 2021), a phylogenetic tree of the five most closely-related families, namely Calcarisporiaceae, Clavicipitaceae, Ophiocordycipitaceae, Cordycipitaceae, and Polycephalomycetaceae was inferred (Fig. 2 The respective partition best-fit models were: TIM + F + I + G4 for LSU, TVM + F + I + G4 for ITS, K2P + I + G4 for SSU, TIM3 + F + I + G4 for TEF-1α and RPB1, and TIM + F + I + G4 for RPB2. The GTR + I + G model of nucleotide substitution was deemed as the most suitable for Bayesian inference analysis. Six simultaneous Markov chains were run for 1,120,000 generations, and trees were sampled every 1,000th generation, thus obtaining 11,201 trees. The topology of the five-family tree reflected that of the Hypocreales backbone tree ( Fig. 1) in terms of branching order. The topologies of family-level IQ maximum likelihood and Bayesian inference analyses were similar. Figure 2 illustrates the IQ maximum likelihood tree. All families were monophyletic with nearly maximum statistical support. The newly established family Polycephalomycetaceae was sister to Ophiocordycipitaceae (96% ML/1.00 PP).

Polycephalomycetaceae and genus-level trees
The representative taxa in Polycephalomycetaceae were included to further explore the phylogenetic placement and relationships between and within Perennicordyceps, Pleurocordyceps, and Polycephalomyces (Fig. 3). The alignment contained members of the newly-proposed family and comprised of 90 taxa (Table 3). Tolypocladium ophioglossoides      The three genera were monophyletic with maximum support (100% ML/1.00 PP). The Pleurocordyceps and Perennicordyceps were sister taxa and this relationship also had maximum support. Based on phylogenetic analyses, five new species and one new combination belonging to Pleurocordyceps are introduced. Pleurocordyceps nutansis had a sister relationship with Pl. sinensis by maximum support (100% ML/1.00 PP). Pleurocordyceps heilongtanensis had a sister relationship with the clade formed by Pl. sinensis and Pl. nutansis with a 99% ML/0.99 PP support (Fig. 2). Pleurocordyceps vitellina was clustered with Pl. agarica as a separate clade      (Fig. 3). The new collection (GACP 21-WFKQ03) parasitized a new host (Ophiocordyceps multibrachiata) and clustered with Po. formosus with maximum support (100% ML/1.00 PP) (Fig. 3). Hence, the new collection was identified as Polycephalomyces formosus. A new species, Po. albiramus (GACP 21-XS08), with its isolate (GACPCC 21-XS08), was sister to Po. formosus, type species. Three additional trees, one for each genus of Polycephalomycetaceae, were also generated ( Fig. 4 for Polycephalomyces; Fig. 11 for Perennicordyceps; Fig. 16 for Pleurocordyceps). Cordyceps pleuricapitata strains also formed a monophyletic clade (100% ML/1.00 PP). The clade was sister to Polycephalomyces and this relationship had maximum statistical support (100% ML/1.00 PP) (Fig. 2, Fig. 3).
The new Po. albiramus strain clustered as sister to Po. formosus but this relationship is not strongly supported and is likely to change as the genus becomes populated with more species. Cordyceps pleuricapitata branches sister to Polycephalomyces with maximum support (Figs. 2, 3, Polycephalomyces clade). Polycephalomyces albiramus differs from Po. formosus in that it produces numerous branched synnemata without a fertile head, shorter conidiophores, and smaller conidia (see notes for Po. albiramus).
All GenBank accession numbers for Polycephalomyces are listed in Supplementary Table 3, excluding ITS: MH859198 (Po. tomentosus, strain CBS 653.68), ITS: AB208109 (Po. tomentosus, strain JCM 12445), and ITS: AJ786598 (Po. ramosus, strain 3078.H). These DNA sequences did not pass our quality control check (blast search indicated incorrect identification). All described species of Polycephalomyces are listed in Supplementary  Table 4.
The ecological and economic significance of Polycephalomyces: Although lacking chemical investigation, this genus has high potential in terms of biotechnological value. Po. formosus and Po. cylindrosporus have a broad range of insect and fungal hosts (Mains 1948;Samson et al. 1981;Wang et al. 2021).
The new collections differ by 1 bp in the untrimmed ITS (488 bp), 2 bp in LSU (749 bp), 2 bp in TEF-1α (734 bp), 4 bp in RPB1 (640 bp), and 107 bp in RPB2 (608 bp) from the holotype of Perennicordyceps elaphomyceticola. The new collections differ by 1 bp in ITS, 2 bp in LSU, 1 bp in SSU, 2 bp in TEF-1α, 4 bp in RPB1, and 4 bp in RPB2, from the other strains of Pe. elaphomyceticola provided with the holotype in the same publication. The RPB2 sequence of the ex-type (NTUCC 17-021) is clearly distinct from the other strains (NTUCC 17-022, NTUCC 17-023) and the new collections from China. In this case, the RPB2 gene of Fig. 11 Phylogenetic tree of Perennicordyceps based on concatenation of LSU, SSU, ITS, TEF-1α, RPB1 and RPB2 sequence data. The tree was generated from an alignment of 5214 sites and 30 taxa. The phylogeny was inferred using IQ-tree. Values at the nodes represent IQ-tree bootstrap support and posterior probabilities, in this order. Only bootstrap values greater than 75% and Bayesian posterior probabilities over 0.90 are shown. The new species are indicated in blue font the ex-type (NTUCC 17-021) needs to be resequenced for clarification.
Moreover, two new collections were fungicolous on Ophiocordyceps sp. (Fig. 13). Ophiocordyceps sp. is a new host for Perennicordyceps elaphomyceticola, which broadens the range of hosts.
Notes: The new species is sister to Pe. paracuboidea with nearly maximum support (100% ML/1.00 PP, Fig. 2; 99% ML/1.00 PP, Fig. 3; 97% ML/1.00 PP, Fig. 11). Perennicordyceps lutea differs from other species of Perennicordyceps in having one-type and longer phialides, as well as, larger globose to ellipsoid conidia. Parasitic on insects or fungi. Sexual morph: Stromata solitary to numerous, fleshy, cylindrical, unbranched, or branched, reddish brown to yellow, stipitate, growing from the head or the whole body of the host. Stipes pigment, cylindrical, sterile tip curved or twisted, with or without a fertile part. Fertile parts capitate at the apex or laterally on the stipe, white to yellow or brown. Perithecia immersed in an apical or subapical pulvinate cushion, scattered to gregarious, pyriform, or ovoid, with protruding ostioles. Asci cylindrical, hyaline, with apical cap and thick walled. Ascospores filiform, cylindrical, hyaline, breaking into secondary spores. Secondary spores cylindrical, hyaline, of nearly equal length (adapted from Matočec et al. 2014;Xiao et al. 2018;Wang et al. 2021). Asexual morph: Hyphomycetous. Synnemata absent or present, white to yellow, solitary to caespitose, stipitate, unbranched or branched, clavate or spatulate or cylindrical, with or without fertile head covered by conidia masses. Stipes clavate or spatulate or cylindrical, white to yellowish, with or without fertile head at the apex or below the apex. Conidial masses absent or present, yellowish to yellow, on the surface of colony, on the host or the surface of the fertile part. Fertile parts absent or present, yellowish to yellow, awl-shaped to cylindrical to globose, at the apex or below the apex. Conidiophores simple or with several phialides, unbranched or branched, hyaline. Metulae absent or present, smooth-walled, hyaline. Phialides (conidiogenous cells) usually two types, sometimes only one type observed on the fresh specimen, verticillately branching, α-phialides verticillate and acropleurogenous on conidiophores, cylindrical to subulate at the base, gradually tapering into a long neck: β-phialides acropleurogenous and solitary, narrow lageniform or subulate, tapering abruptly from the base to the apex. Conidia one-celled, hyaline, smooth-walled, normally two types in culture, rarely two types on fresh specimen, α-conidia globose to subglobose or ellipsoidal, forming conidial masses from the fertile head; β-conidia fusiform, produced along stipe as well as on the mycelium surface of the colony, single, often in chains (adapted from Matočec et al. 2014;Xiao et al. 2018;Wang et al. 2021). Basionym: Paecilomyces sinensis Q.T. Chen et al., Acta Mycol. Sin. 3: 25 (1984).
Ecological and economic significance Extracts of Pl. nipponica mycelia are potential sources of natural antioxidant, antibacterial, anti-breast cancer, and antimicrobial compounds, which can be used in the pharmaceutical sector (Sangdee et al. 2017;Somsila et al. 2018). Pleurocordyceps phaothaiensis has shown antibacterial and anti-inflammatory activities in vitro (Sonyot et al. 2020).
Material examined: CHINA, Guizhou Province, Zunyi City, Wuchuan County, parasitic on larva of Lepidoptera, on the leaf litter, 23 October 2019, Yu Yang, new host: GACP 19-2301. Notes: In the phylogenetic tree, the new strains group with other sequences of Pl. sinensis forming a Pl. sinensis clade (Fig. 16). Moreover, the morphological characters are the same as Pl. sinensis. Hence, the two newly isolated strains are identified as Pl. sinensis based on morphology and phylogenetic analyses. However, the host of these strains differs from the holotype of Pl. sinensis, which parasitizes O. sinensis. The two strains parasitize larvae of Lepidoptera (Fig. 17: GACP 19-2301, Fig. 18: MFLU 21-0269), O. neoacicularis (Fig. 19:124 MFLU 21-0268), O. crinalis (Fig. 20: GACP 20-0865) and O. barnesii (Fig. 21: GACP 20-2304), all of which are new hosts of Pl. sinensis. Specimens were collected from Guizhou and Yunnan, China. Hence, we introduce three new hosts for Pl. sinensis, illustrating its broad host range. This study also shows the geographic distribution of Pl. sinensis, which keeps expanding having now been found in the neighboring provinces of Yunnan, Sichuan and Guizhou.
Material examined: CHINA, Guizhou Province, Chishui City, parasitic on O. crinalis (Ophiocordycipitaceae), on the leaf litter, 30 September 2019, Yu Yang, Notes: The new collection grouped with the holotype of Pl. sinensis and has the same morphological features. Thus, we identified it as Pl. sinensis. Ophiocordyceps crinalis is a new host species of Pl. sinensis and will be discussed in a future study.
In the phylogenetic tree, the new species is sister to Pl. lianzhouensis with high support (Figs. 2, 3: 100 ML/1.00 PP; Fig. 16: 96% ML/1.00 PP). The new species, Pl. parvicapitata, differs from other species of Pleurocordyceps in that it parasitizes Elaphomyces sp. (Elaphomycetaceae, Eurotiales) and produces capitate fertile heads, long stipes, and shorter secondary ascospores (Table 8). The asexual morph of Pl. parvicapitata is characterized by parasitizing both Pe. elaphomyceticola and Elaphomyces sp., 2-6 clustered phialides (Table 7). Only one type of phialides has been discovered from a fresh specimen. Furthermore, the new species, Pl. parvicapitata, is the first report for taxa parasitic on Perennicordyceps among all family species, contributing to a deeper understanding of the physiology, evolution, and taxonomy of these groups.
Index Fungorum number: IF559474; Facesoffungi number: FoF 10740 Etymology: The species name refers to the lanceolate synnemata both on fresh specimens and culture.
Notes: Pleurocordyceps heilongtanensis is branching off the clade formed by Pl. sinensis, Po. ramosus and Po. tomentosus in the phylogenetic tree with nearly maximum support ( Fig. 16: 99% ML/1.00 PP). More data is needed for Po. ramosus and Po. tomentosus. Pleurocordyceps heilongtanensis is distinct from Pl. sinensis in that it produces smaller α-phialides and subglobose to ovoid β-conidia (Table 7). Thus, we introduce Pl. heilongtanensis as a new species and identify the host as Ophiocordyceps sp. Etymology: The species name refers to the yolk yellow color of conidial masses.
Notes: The new collections (MFLU 21-0276, GACP 20-2306) cluster with other Pl. aurantiacus sequences with 100% ML/1.00 PP; Fig. 3: 97% ML/0.99 PP (Fig. 16) support. Morphology on PDA is the same as Pl. aurantiacus. Pleurocordyceps aurantiacus was parasitic on O. neobuquetii, which parasitized a queen of green the tree ant (Formicidae, Oecophylla smaragdina) in a tree hollow, from Phayao Province, Thailand. The host of the holotype was first reported as Cossidae larvae (Lepidoptera) (Xiao et al. 2018) and on beetle larvae (Coleoptera) in this study. The β-phialides and β-conidia were observed with synnemata and stromata on the holotype, while α-phialides and α-conidia were observed with the colony on the fresh specimen. Thus, O. neouguetii is the new host for Pl. aurantiacus. Colony obverse and reverse on PDA. m, n Synnemata. o β-phialides. p α-phialides with slime conidia masses. q α-conidia. r β-conidia. Scale Bars: d, e, k, l = 1 cm, f, m, n = 1 mm, g, o, p = 50 µm, h = 20 µm, i = 10 µm, j, q, r = 5 µm r Colony obverse and reverse on PDA. s-u Synnemata in culture. v Conidiophores. w α-phialides. x α-conida. y β-phialides. z β-conidia. Scale Bars: r = 1 cm, s = 5 mm, f, t = 1 mm, g, i = 500 µm, j, k, o, u = 200 µm, h, l, v, y = 20 µm, p, w = 10 µm, m, n, q, x, z = 5 µm five families. In all analyses and irrespective of the dataset used, the three genera always grouped together with nearly maximum statistical support. Clustering of the three genera has been consistent with previous studies (Wang et al. 2012(Wang et al. , 2015aKepler et al. 2013;Zhong et al. 2016;Crous et al. 2017;Xiao et al. 2018;Poinar and Vega 2020). These results clearly illustrate the monophyly of the new family. However, the position of Polycephalomycetaceae has not been stable across studies. Previously, members of the family have placed as sister to either Ophiocordycipitaceae (Matočec et al. 2014;Wang et al. 2015b;Crous et al. 2017;Xiao et al. 2018) or Clavicipitaceae (Wang et al. 2012;Zhong et al. 2016). Using the same six genetic marker dataset to infer phylogenies with IQ-tree and BI resulted in Polycephalomycetaceae grouped with Ophiocordycipitaceae in each of the order-level and five families-level trees. The same placement was recovered in the order level tree inferred without the ITS data. Topology testing indicated that neither hypothesis could be rejected. Therefore, the deeper phylogenetic placement of the new family remains unclear. Future studies using an expanded gene repertoire and additional taxa are needed to resolve this question.
We regard Cordyceps pleuricapitata as incertae sedis in Polycephalomycetaceae. The paratype of C. pleuricapitata lacks molecular data, and the two strains (NBRC 100745, NBRC 100746) named C. pleuricapitata for which there are molecular data lack morphological information. Hence, even though the two strains grouped with Polycephalomyces sp., it is not possible to clarify the precise position of C. pleuricapitata and its classification at this time.
The new family has three genera, Polycephalomyces, Perennicordyceps and Pleurocordyceps. Perennicordyceps and Pleurocordyceps were segregated from Polycephalomyces based on morphology and phylogenetic analyses (Matočec et al. 2014;Wang et al. 2021). The three genera seperately form monophyletic clades within Polycephalomycetaceae. Each genus consists of more than one species and most species have more than one strain. The stable phylogenetic placement, monophyly and strong statistical support reinforce the generic rank of these taxa within the new family. Polycephalomyces species share a similar morphology with Perennicordyceps and Pleurocordyceps, producing acremonium-or hirsutella-like or phialides. Polycephalomyces is characterized by oblong-to-cylindrical conidia (Kobayasi 1941;Wang et al. 2021). Perennicordyceps and Pleurocordyceps species share similar morphology in having stipitate stromata, ovoid to flask-shaped perithecia, a thick peridium, cylindrical asci, filiform ascospores, and cylindrical to globose secondary spores (Matočec et al. 2014;Wang et al. 2020). Pleurocordyceps species are distinct from Perennicordyceps in that they produce capitate stromata and superficial perithecia (Matočec et al. 2014;Wang et al. 2020). Most Perennicordyceps and Pleurocordyceps species produce two types of conidia, with the exception of Pe. ryogamiensis and Pl. lianzhouensis (Ban et al. 2009;Wang et al. 2014).
Herein, we describe a new species Po. albiramus, enriching the diversity of the genus. The new species parasitizes Gryllotalpa sp. (Orthoptera, Gryllotalpidae) and produces cylindrical-to-oval conidia on fresh specimens and in pure culture. Six additional species are included in this genus (Table 1). Polycephalomyces baltica (Poinar and Vega 2020), Po. cylindrosporus (Matočec et al. 2014), Po. ditmarii (Van Vooren and Audibert 2005) and Po. paludosus produce a single type of conidia (Mains 1948); Polycephalomyces ramosus with two types of conidia (Seifert 1985;Bischoff et al. 2003); Polycephalomyces tomentosus with three types of conidia (Seifert 1985). However, these species either lack molecular data, or their updated strains descriptions do not match those of the protologue (Wang et al. 2021). Although these six species have been clarified under the genus Polycephalomyces, additional morphologic and phylogenetic work is required to clarify their taxonomic status.
A new collection of the type species Po. formosus from China was described, which has one-type conidia. This collection was growing on O. multibrachiata, which is a new host record. Polycephalomyces formosus has been found to parasitize stromata of O. barnesii (original host name: C. barnesii), and O. cantharelloides (original host name: C. cantharelloides) on larvae of Coleoptera (Kobayasi 1941;Seifert 1985). The new collection adds to the host range of this fungus.
Pleurocordyceps sinensis occurred on Ophiocordyceps species from the same region. Specifically, Pl. sinensis was collected from the southern neighboring provinces of Yunnan and Guizhou. In Yunnan the species parasitized O. neoacicularis, while in Guizhou it parasitized O. barnesii and O. crinalis. In Yunnan, Pl. sinensis also parasitized Lepidoptera larvae. Pleurocordyceps sinensis was initially reported as parasitic on O. sinensis in Tibet (Chen et al. 1984;Wang et al. 2012). Thus Pl. sinensis is a parasite that is not restricted to a specific Ophiocordyceps host from a specific region. Five species of Pleurocordyceps were collected from the same region in Yunnan Province, while two were collected from the same region in Guizhou Province. Two new species Pl. heilongtanensis and Pe. lutea are found on Ophiocordyceps sp. and O. sinensis, respectively.
Thailand and southern China are areas of high fungal biodiversity . In this study, seven new species and three new records were identified from the two countries. The high diversity that was uncovered suggests that further collections will result in numerous new taxa being discovered (Hyde et al. 2020a, b). The discovery of new taxa populated a distinct group of clavicipitoid fungi and resulted in the establishment of the novel family Polycephalomycetaceae. This indicates that we are far from fully understanding the classification of fungi, even in the relatively well-studied Hypocreales. Hence, future studies should focus on obtaining additional collections to fully understand the diversity and evolution of clavicipitoid fungi in Hypocreales.