Front. Microbiol. Frontiers in Microbiology Front. Microbiol. 1664-302X Frontiers Media S.A. 10.3389/fmicb.2023.1256967 Microbiology Original Research Four novel Pleurocordyceps (Polycephalomycetaceae) species from China Xiao Yuan-Pin 1 † Yang Yu 1 2 3 † Jayawardena Ruvishika S. 2 3 Gentekaki Eleni 4 Peng Xing-Can 2 3 5 Luo Zong-Long 6 Lu Yong-Zhong 1 5 * 1School of Food and Pharmaceutical Engineering, Guizhou Institute of Technology, Guiyang, China 2Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, Thailand 3School of Science, Mae Fah Luang University, Chiang Rai, Thailand 4University of Nicosia School of Veterinary Medicine, Nicosia, Cyprus 5Engineering and Research Center for Southwest Bio-Pharmaceutical Resources of National Education Ministry of China, Guizhou University, Guiyang, China 6College of Agriculture and Biological Sciences, Dali University, Dali, China

Edited by: Zhou Shi, Gladstone Institutes, United States

Reviewed by: Samantha Chandranath Karunarathna, Qujing Normal University, China

Yang Liu, Dana–Farber Cancer Institute, United States

 *Correspondence: Yong-Zhong Lu yzlu@git.edu.cn

†These authors have contributed equally to this work

 10 01 2024 2023 14 1256967 12 07 2023 08 12 2023 Copyright © 2024 Xiao, Yang, Jayawardena, Gentekaki, Peng, Luo and Lu. 2024 Xiao, Yang, Jayawardena, Gentekaki, Peng, Luo and Lu

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Entomopathogenic fungi comprise an ecologically important group of specialized pathogens infecting other fungi, invertebrates, and plants. These fungi are species-rich with high diversity and broad distribution worldwide. The majority of entomopathogenic fungi belong to clavicipitoids, which consist of the hypocrealean families, Clavicipitaceae, Cordycipitaceae, Ophiocordycipitaceae, and Polycephalomycetaceae. The latter is a newly established entomopathogenic family that recently separated from the family Ophiocordycipitaceae to accommodate the genera, Perennicordyceps, Pleurocordyceps, and Polycephalomyces. In recent years, Polycephalomycetaceae has been enriched with parasitic and hyperparasitic fungi. With 16 species spread across China, Ecuador, Japan, and Thailand, Pleurocordyceps is the most speciose genus in the family. In this study, we expand the number of taxa in the genus by introducing four new Pleurocordyceps species from China, namely, P. clavisynnema, P. multisynnema, P. neoagarica, and P. sanduensis. We provide detailed descriptions and illustrations and infer genus-level phylogenies based on a combined 6-loci gene sequence dataset comprising the 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). This study contributes to knowledge with regard to the diversity of Pleurocordyceps specifically and entomopathogenic Hypocreales more broadly.

 entomopathogenic fungi morphology Polycephalomycetaceae phylogeny taxonomy section-at-acceptance Microbial Symbioses Introduction

Insect pathogenic fungi, also known as entomopathogenic fungi, comprise a group of over 2,000 species spanning 90 genera (Saltamachia and Araujo, 2020). The phylogenetic diversity of entomopathogenic fungi is notable, with the majority belonging to Hypocreales, the largest group of plant and insect pathogens in Sordariomycetes (Sung et al., 2007; Maharachchikumbura et al., 2016; Wijayawardene et al., 2018). Within Hypocreales, the families Clavicipitaceae, Cordycipitaceae, Ophiocordycipitaceae, and Polycephalomycetaceae are collectively known as the clavicipitoid fungi and contain the majority of known insect pathogens (Hyde et al., 2020; Wei et al., 2020; Wijayawardene et al., 2020; Huang et al., 2021; Xiao et al., 2023). Some species are well known in the fields of agriculture and related industries, including Beauveria bassiana (biological control agent), Cordyceps militaris (medicinal), Metarhizium anisopliae (biological control agent), and Ophiocordyceps sinensis (medicinal) (Zimmermann, 2007; Acuña Jiménez et al., 2015; Li et al., 2020; Eiamthaworn et al., 2022). Thus, entomopathogenic fungi have gained the attention of researchers as a crucial fungal resource (Fernández-Grandon et al., 2020; Sharma et al., 2020; Sobczak et al., 2020; Zha et al., 2021).

The taxonomy of entomopathogenic fungi has undergone substantial changes since the advent of the molecular era (Tasanathai et al., 2016; Dong et al., 2022). Chaverri et al. (2005) initiated this molecular exploration by providing LSU, TEF, and RPB1 data for Polycephalomyces formosus and Polycephalomyces ramosopulvinatus (current name: Pleurocordyceps ramosopulvinata). Ban et al. (2009) used a 504-base-pair LSU fragment, but it fell short in resolving deep fungal nodes (Kepler et al., 2013). Different loci were selected for the analysis of novel species, with Wang et al. (2014) using a 4-loci (SSU, LSU, TEF, and RPB1), Wang et al. (2015b) using a 5-loci (SSU, LSU, TEF, RPB1, and RPB2), and Wang et al. (2015a) and Xiao et al. (2018) utilizing a 6-loci (ITS, SSU, LSU, TEF, and RPB1, and RPB2). The phylogenetic placement of Polycephalomyces or the segregation of new genera from Polycephalomyces was analyzed using both 5-loci (SSU, LSU, TEF, RPB1, and RPB2) and 6-loci (ITS, SSU, LSU, TEF, RPB1, and RPB2) (Kepler et al., 2013; Matočec et al., 2014; Wang et al., 2021). Building on this molecular groundwork, Xiao et al. (2023) established a new family, Polycephalomycetaceae, accommodating three genera (Perennicordyceps, Pleurocordyceps, and Polycephalomyces) and comprising 28 species using 6 loci (ITS, SSU, LSU, TEF, RPB1, and RPB2).

Over the past decade, a multitude of new species have been described in the family Polycephalomycetaceae, including those documented by Kepler et al. (2012), Wang et al. (2015a,b), and Yang et al. (2020), contributing to a deeper understanding of its classification. Recent studies by Wei et al. (2022) and Xiao et al. (2023) have introduced additional new species, sparking renewed interest in the taxonomy of the family. The sexual morph of Polycephalomycetaceae is distinguished by producing superficial or immersed ascomata with a stipe, three layers of peridium, narrowly cylindrical asci, multiseptate ascospores, and short cylindrical part spores (Matočec et al., 2014; Wang et al., 2021; Xiao et al., 2023). Its asexual morphs have congregated mycelia on the surface of the host, light-colored synnemata with stipules, divergent conidiophores, and one or both types of phialides and conidia (Matočec et al., 2014; Wang et al., 2021; Xiao et al., 2023). Most species in Polycephalomycetaceae are found in tropical and subtropical regions, with fewer taxa found in temperate regions (Van Vooren and Audibert, 2005; Wang et al., 2012, 2015a; Matočec et al., 2014; Xiao et al., 2018, 2023). A high diversity of polycephalomycetous fungi has been found in China and Japan (Kobayasi, 1939, 1941; Kobayasi and Shimizu, 1982; Chen et al., 1984; Wang et al., 2012, 2014, 2015a,b, 2021; Kepler et al., 2013; Quandt et al., 2014; Yang et al., 2020; Xiao et al., 2023).

With 16 species, Pleurocordyceps is the most speciose genus in the family Polycephalomycetaceae (Wang et al., 2021; Xiao et al., 2023). Pleurocordyceps was established by Wang et al. (2021) with the type species, P. sinensis, which was found on Ophiocordyceps sinensis (Chen et al., 1984). Pleurocordyceps is distinguished from closely related genera by its lateral fertile pulvinate stromata near the tip of the sexual morph and its two types of phialides and conidia in the asexual morph (Wang et al., 2021; Xiao et al., 2023). Wang et al. (2021) provided a key to the 10 accepted Pleurocordyceps species (Wang et al., 2021; Xiao et al., 2023). The insect host orders associated with Pleurocordyceps sp. comprise Coleoptera, Hymenoptera, Hemiptera, Lepidoptera, Orthoptera, and Homoptera (Kobayasi, 1939; Kobayasi and Shimizu, 1982; Bischoff et al., 2003; Ban et al., 2009; Wang et al., 2012, 2015a,b; Crous et al., 2017; Xiao et al., 2018; Poinar and Vega, 2020). In addition to parasitizing insects, most species in the genus are also parasites of fungi (Kobayasi, 1941; Seifert, 1985; Bischoff et al., 2003; Ban et al., 2009; Wang et al., 2015a; Xiao et al., 2023). In recent years, Ophiocordyceps sp. has been frequently reported as the host of Polycephalomyces-like species (Sun et al., 2019; Xiao et al., 2023). Specifically, Pleurocordyceps agarica, P. aurantiacus, P. lianzhouensis, P. sinensis, and P. yunnanensis are parasites on Ophiocordyceps sp. and insects (Chen et al., 1984; Wang et al., 2012, 2015a,b, 2021; Xiao et al., 2018). In general, Pleurocordyceps spp. exhibit significant potential for producing a diverse range of secondary metabolites. For instance, Pleurocordyceps nipponicus and P. phaothaiensis contain natural antioxidant, antibacterial, antitumorigenic, anti-inflammatory, and antimicrobial compounds (Sangdee et al., 2017; Somsila et al., 2018; Sonyot et al., 2020). Gokhale et al. (2020) reported that the secondary metabolites of P. sinensis have antibacterial potential. However, there are noticeable gaps in critical areas, such as chemistry, industry, and ecology of Pleurocordyceps species. Thus, there is a compelling need for further research to explore the wide array of capabilities and applications within Pleurocordyceps.

In China, there are records of nine Pleurocordyceps species, along with more than 200 taxa of clavicipitoid fungi that have been found in the country (Wang et al., 2012, 2014, 2015a,b; Liang et al., 2016; Yang et al., 2020; Xiao et al., 2023). In this study, we introduce four new species of Pleurocordyceps, namely, P. clavisynnema, P. multisynnema, P. neoagarica, and P. sanduensis. We provide a detailed morphological description along with phylogenetic analyses using a combined 6-loci gene region (ITS, SSU, LSU, tef-1α, rpb1, and rpb2).

 Materials and methods Sample collection, isolation, and morphological studies

Fresh specimens, comprising a total of eight, were collected from soil in Anhui and Guizhou provinces, China. The samples were transported in plastic boxes to the laboratory, and pertinent metadata (location, longitude, and latitude) were recorded. The fruiting bodies were examined using a stereomicroscope (SMZ 745 and SMZ 800N, Nikon, Tokyo, Japan) and free-hand sections were obtained for analysis. Micromorphological features such as synnemata, conidiophores, phialides, and conidia were captured using a Nikon DS-Ri2 digital camera connected to a Nikon ECLIPSE microscope (Tokyo, Japan). The strains were obtained from fresh tissue by removing a small piece of mycelium from the host, which was then transferred with a sterile needle onto PDA plates and incubated at 25°C. The pure culture was stored in the Guizhou Culture Collection, China (GZCC). The specimens were deposited at the Guizhou Institute of Technology Herbarium (Herb. GZLG). The guidelines of the Facesoffungi database (https://www.indexfungorum.org) were followed to obtain Index Fungorum numbers, as outlined by Jayasiri et al. (2015). The morphological structures were measured using Tarosoft (R) v.0.9.7 Image Frame Work, and the photographic plates were processed using Adobe Photoshop CC 2022 (Adobe Systems, USA).

 DNA extraction, PCR amplification, and sequencing

Total DNA was extracted from fruiting bodies and cultures using the Fungal DNA MiniKit (Biotech, USA), following the manufacturer's instructions. Internal transcribed spacer gene region (ITS), small subunit ribosomal RNA gene region (SSU), large subunit rRNA gene region (LSU), RNA polymerase II largest subunit gene region (rpb1), RNA polymerase II second largest subunit (rpb2), and translation elongation factor 1-alpha gene region (tef-1α) gene amplifications were performed using the ITS5/ITS4, NS1/NS4, LR0R/LR5, CRPB1A/RPB1Cr, fRPB2-5F/fRPB2-7Cr, and 983F/2218R primers, respectively (Vilgalys and Hester, 1990; White et al., 1990; Hopple and Vilgalys, 1999; Castlebury et al., 2004; Sung et al., 2007). Previous studies have demonstrated that the use of these six genetic loci optimally resolves the phylogenetic placement of the species Pleurocordyceps (Xiao et al., 2018, 2023; Wang et al., 2021; Wei et al., 2022). The nuclear gene amplification reactions followed the protocol outlined by Yang et al. (2021). PCR products were sent to Tsingke Biotechnology for sequencing (Chongqing, China). All newly generated sequences were uploaded to GenBank, and accession numbers were assigned (Table 1).

Accession numbers of DNA sequences used in the phylogenetic analyses.

Taxon Strain GenBank accessions References
ITS SSU LSU rpb1 rpb2 tef-1α
Pleurocordyceps agarica YHHPA1305 KP276651 KP276655 - KP276663 KP276667 KP276659 Wang et al., 2015b
P. agarica YHCPA1307 KP276654 KP276658 - KP276666 KP276670 KP276662 Wang et al., 2015b
P. aurantiacus MFLUCC 17-2113 MG136916 MG136904 MG136910 MG136866 MG136870 MG136875 Xiao et al., 2018
P. aurantiacus MFLU 17-1394 MG136918 MG136906 MG136912 MG136867 MG136872 MG136876 Xiao et al., 2018
P. clavisynnema GZLG 23-102 OQ968788 - OQ968796 - - OQ982009 This study
P. clavisynnema GZCC 22-2042 OQ968789 OQ968805 OQ968797 OQ981998 OQ982004 OQ982008 This study
P. formosus ARSEF1424 KF049661 KF049615 KF049634 KF049651 KF049671 KF049689 Kepler et al., 2013
P. formosus MFLU 18-0162 MK863250 MK863043 MK863050 MK860188 - - Unpublished
P. heilongtanensis KUMCC 3008 OQ172091 OQ172111 OQ172063 OQ459759 OQ459805 OQ459731 Xiao et al., 2023
P. kanzashianus - AB027371 AB027371 AB027325 - - - Nikoh and Fukatsu, 2000
P. lanceolatus GACPCC 17-2005 OQ172077 OQ172109 OQ172047 OQ459755 OQ459801 OQ459727 Xiao et al., 2023
P. lanceolatus GACP 17-2004 OQ172076 OQ172110 OQ172046 OQ459754 OQ459800 OQ459726 Xiao et al., 2023
P. lianzhouensis HIMGD20918 EU149921 KF226245 KF226246 KF226247 - KF226248 Zhang et al., 2007
P. lianzhouensis GIMYY9603 EU149922 KF226249 KF226250 KF226251 - KF226252 Zhang et al., 2007
P. marginaliradians MFLU 17-1582 MG136920 MG136908 MG136914 MG136869 MG271931 MG136878 Xiao et al., 2018
P. multisynnema GZLG 23-101 OQ968792 OQ968802 OQ968800 - OQ982002 - This study
P. multisynnema GZCC 22-2041 OQ968793 OQ968803 OQ968801 OQ981997 OQ982003 OQ982010 This study
P. neoagarica GZLG 23-103 OQ968790 - OQ968795 - - - This study
P. neoagarica GZCC 22-2043 OQ968791 OQ968804 OQ968794 OQ981996 OQ981999 OQ982007 This study
P. nipponicus NHJ 4268 KF049657 KF049621 KF049639 MF416676 KF049676 MF416517 Kepler et al., 2013
P. nipponicus BCC 1682 KF049664 KF049620 KF049638 - - KF049694 Kepler et al., 2013
P. nipponicus NBRC 101408 JN943303 JN941751 JN941390 JN992485 - - Schoch et al., 2012
P. nipponicus BCC 2325 KF049665 KF049622 KF049640 KF049655 KF049677 KF049696 Kepler et al., 2013
P. nutansis GACP 19-1906 OQ172079 OQ172117 OQ172049 OQ459763 OQ459809 OQ459737 Xiao et al., 2023
P. nutansis MFLU 21-0275 OQ172073 OQ172119 OQ172048 OQ459765 OQ459811 OQ459739 Xiao et al., 2023
P. onorei BRA CR23902 KU898841 - - - - - Crous et al., 2017
P. onorei BRA CR23904 KU898843 - - - - - Crous et al., 2017
P. phaothaiensis BCC84557 MF959734 - MF959738 MF959746 - MF959741 Crous et al., 2017
P. phaothaiensis BCC84553 MF959733 - MF959737 MF959745 - MF959742 Crous et al., 2017
P. ramosus RUTPP - - AY259543 - - - Bischoff et al., 2003
P. ramosus like NBRC 101760 MN586827 MN586818 MN586836 MN598042 MN598060 MN598051 Wang et al., 2021
P. ramosus like NBRC 109984 MN586828 MN586819 MN586837 MN598043 - MN598052 Wang et al., 2021
P. ramosus like NBRC 109985 MN586829 MN586820 MN586838 MN598044 - MN598053 Wang et al., 2021
P. ramosopulvinatus EFCC 5566 KF049658 - KF049627 KF049645 - KF049682 Kepler et al., 2013
P. ramosopulvinatus SU 65 - - DQ118742 DQ127244 - DQ118753 Chaverri et al., 2005
P. ramosopulvinatus - AB027372 AB027326 - - - - Nikoh and Fukatsu, 2000
P. sinensis CGMCC 3.19069 MH459160 MH454346 - - - - Sun et al., 2019
P. sinensis CN 80 2 HQ832884 HQ832887 HQ832886 HQ832888 HQ832889 HQ832890 Wang et al., 2012
P. sinensis HMAS 43720 NR 119928 - NG 042573 - - KF049697 Wang et al., 2012
P. sanduensis GZLG 23-104 OQ968786 - OQ968798 - OQ982000 OQ982005 This study
P. sanduensis GZCC 22-2044 OQ968787 OQ968806 OQ968799 OQ981995 OQ982001 OQ982006 This study
P. tomentosus BL4 KF049666 KF049623 KF049641 KF049656 KF049678 KF049697 Kepler et al., 2013
P. vitellina KUMCC 3005 OQ172088 - OQ172060 OQ459756 OQ459802 OQ459728 Xiao et al., 2023
P. vitellina KUMCC 3006 OQ172089 - OQ172061 OQ459757 OQ459803 OQ459729 Xiao et al., 2023
P. vitellina KUMCC 3007 OQ172090 - OQ172062 OQ459758 OQ459804 OQ459730 Xiao et al., 2023
P. yunnanensis YHCPY1005 KF977848 KF977848 KF977848 KF977852 KF977854 KF977850 Wang et al., 2015a
P. yunnanensis YHHPY1006 KF977849 KF977849 KF977849 KF977853 KF977855 KF977851 Wang et al., 2015a
P. sp. BCC 2637 KF049663 KF049619 KF049637 - KF049675 KF049693 Kepler et al., 2013
P. sp. JB07 08 16 08 KF049662 KF049616 KF049635 KF049652 KF049672 KF049690 Kepler et al., 2013
P. sp. JB07 08 17 07b - KF049617 - KF049653 KF049673 KF049691 Kepler et al., 2013
P. sp. NBRC 109987 - - AB925983 - - - Wang et al., 2021
P. sp. NBRC 109988 - - AB925984 - - - Wang et al., 2021
P. sp. NBRC 109990 - - AB925968 - - - Wang et al., 2021
P. sp. NBRC 110224 - - AB925969 - - - Wang et al., 2021
P. sp. GIMCC 3 570 - JX006097 JX006098 JX006101 - JX006100 Wang et al., 2021
Perennicordyceps cuboidea NBRC 101740 JN943321 JN941734 JN941407 JN992468 AB968564 AB968603 Schoch et al., 2012
Pe. cuboidea NBRC 10383 JN943319 JN941735 JN941406 JN992469 AB968563 AB968602 Kepler et al., 2013

Newly generated sequences are indicated in bold. “-” means no data available in GenBank. ARSEF, USDA-ARS Collection of Entomopathogenic Fungal Cultures, Ithaca; BCC, BIOTEC Culture Collection, Khlong Luang, Thailand; CBS, Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands; CGMCC, China General Microbiological Culture Collection Center, Beijing, China; EFCC, Entomopathogenic Fungal Culture Collection, Chuncheon, Korea; GACP, Herbarium of Guizhou University, China; GZCC, Guizhou Culture Collection, Guizhou Academy of Agricultural Sciences, Guiyang, China; KUMCC, Culture collection of Kunming Institute of Botany, Kunming, China; MFLU, Mae Fah Luang University, Thailand; NBRC, Culture Collection Division Biological Resource Center (NBRC) National Institute of Technology and Evaluation.

 Phylogenetic analyses

Using SeqMan, all newly generated sequences were assembled (Clewley, 1995). The reference taxa for phylogenetic analyses were obtained based on the BLAST search results (https://blast.ncbi.nlm.nih.gov/Blast.cgi) against the non-redundant protein sequence database (NRDB) using default parameters and previously published datasets (Table 1). Individual sequences were aligned using MAFFT v.7 (https://mafft.cbrc.jp/alignment/server/) and trimmed with Trimal v 1.4 (Capella-Gutiérrez et al., 2009; Katoh and Standley, 2013). Alignment was manually adjusted using BioEdit where needed (Hall, 1999). Maximum likelihood (ML) and Bayesian inference (BI) were used to infer phylogenies from a combined six-genetic marker dataset. Outgroup taxa were chosen as Perennicordyceps cuboidea (NBRC 101740) and Pe. cuboidea (NBRC 103836) (Schoch et al., 2012).

The ML phylogeny was inferred using IQ-TREE 2 with partitioned models and 1,000 exhaustive bootstrap replications (Minh et al., 2020). The model of evolution for each locus was chosen by the built-in ModelFinder tool (Kalyaanamoorthy et al., 2017). The BI analysis was conducted using MCMC sampling and MrBayes version 3.1.2 (Ronquist et al., 2012). The sampling was performed with six simultaneous Markov chains for 1,850,000 generations based on the standard deviation of split frequencies being < 0.01, with trees being sampled every 1,000 generations. The initial 25% of trees were considered as the burn-in phase and were discarded. The posterior probability (PP) was calculated using the remaining trees (Dissanayake et al., 2020). FigTree v.1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/) was used to visualize the ML tree. Based on the guidelines provided by Chethana et al. (2021), Jayawardena et al. (2021), and Maharachchikumbura et al. (2021), novel species descriptions were created.

 Results Phylogenetic analyses

Sequences from 58 taxa representing 24 species of the family Polycephalomycetaceae were obtained from GenBank. The alignment contained 4,791 characteristics, representing 58 taxa. LSU: 847 bp, ITS: 531 bp, SSU: 943 bp, tef-1α: 844 bp, rpb1: 680 bp, and rpb2: 946 bp sequence data, including gaps, were combined in the final alignment. Outgroup taxa included Perennicordyceps cuboidea (NBRC 101740) and Perennicordyceps cuboidea (NBRC 103836). The topologies of ML and BI analyses were nearly congruent. Figure 1 displays that the maximum likelihood bootstrap (MLBS) is higher than 75%. The collections were determined as four new species, namely, Pleurocordyceps clavisynnema, P. multisynnema, P. neoagarica, and P. sanduensis. The phylogenetic placement of the new species is described in detail in the notes section below.

Maximum likelihood phylogenetic tree of 58 taxa and 4,791 sites combining LSU, SSU, ITS, tef-1α, rpb1, and rpb2 sequence data. MLBS higher than 75% and PP >0.90 are denoted near the nodes as MLBS/PP, and the newly generated sequences are in red bold font. The genus clade Pleurocordyceps is highlighted in green, while the outgroup is marked with a light orange background.

 Taxonomy

Pleurocordyceps clavisynnema Y. P. Xiao and Y. Yang sp. nov (Figure 2).

Index Fungorum number: IF900449; Faceoffungi number: FoF 14158

Etymology: Name referring to clavate synnemata.

Holotype: GZLG 23-102

Pleurocordyceps clavisynnema (GZLG 23-102, Holotype). (a, b) Host: Ophiocordyceps neogryllotalpae (c) Upper side of the colony. (d) Synnemata on the culture. (e) Conidiophores. (f, g) α-phialides. (h) β-phialides. (i) α-conidia. (J) β-conidia. Scale bars: (b, c) 3 cm, (d) 0.5 cm, (e) 100 μm, (f–h) 20 μm, (i, j) 3 μm.

Parasitism on Ophiocordyceps neogryllotalpae (Ophiocordycipitaceae, Hypocreales). Sexual morph: Not observed. Asexual morph: Hyphomycetous. Culture characteristics: Colonies on PDA fast-growing, derived from tissue isolation, reaching 3 cm wide in 2 weeks at 25°C, white, and obverse brown. Synnemata emerging after 20 days, clavate or with a mucronate apex, solitary, unbranched, and 2–5 mm long. Fertile head 0.6–2.3 mm wide, yellowish to yellow, emerging on the middle part of the synnemata or on the top, with conidial masses on the surface. Conidial masses brown, slimy. Conidiophore 21–39 μm long (x¯ = 20 μm, n = 40), 2–6 phialides in one. Phialides has two types α-phialides 8.3–14.5 × 0.9–1.7 μm (x¯ = 11.4 × 1.3 μm, n = 40) smooth, hyaline, solitary. β-phialides 12.3–21.6 × 0.8–1.8 μm (x¯ =16.95 × 1.3 μm, n = 40), smooth, hyaline, solitary. α-conidia 1.7–2.6 μm (= 2.15 μm, n = 50) wide, globose, 1-celled, smooth-walled; β-conidia 3.1–4.1 × 1.6–2.2 μm (x¯ = 3.6 × 1.9 μm, n = 50), hyaline, fusiform, 1-celled, smooth.

Material examined: China, Guizhou Province, Qiannan Buyi and Miao Autonomous Prefecture, Sandu Shui Autonomous County. Parasitic on Ophiocordyceps neogryllotalpae (Ophiocordycipitaceae, Hypocreales), in the soil, 10 April 2022, Yu Yang, SD05H (GZLG 23-102, holotype; ex-type living culture, GZCC 22-2042).

Notes: Pleurocordyceps sanduensis is the closest match to our new sample of P. clavisynnema. This is also confirmed by phylogenetic analyses, whereby the two are sister taxa with maximum statistical support (100% ML/1.00 PP; Figure 1). Base pair differences between P. clavisynnema and P. sanduensis are 23/824 in tef-1α, 8/1130 in SSU, 2/678 in rpb1, and 3/1050 in rpb2. Morphologically, P. clavisynnema differs from P. sanduensis by having longer synnemata, larger conidiophore, smaller phialides, and shorter conidia. Hence, this study introduces Pleurocordyceps clavisynnema as a new species based on morphological and phylogenetic analyses.

Pleurocordyceps multisynnema Y. Yang and Y. P. Xiao sp. nov (Figure 3).

Index Fungorum number: IF900451; Faceoffungi number: FoF 14160

Etymology: Name referring to the multiple synnemata of the host and culture.

Holotype: GZLG 23-101

Pleurocordyceps multisynnema (GZLG 23-101, holotype) (a) Habitat. (b) Overview of Pleurocordyceps multisynnema. (c) Synnemata on the host. (d) Host of Paraisaria sp. (e, f) Section of host. (g) β-phialides. (h) α-phialides. (i) α-conidia and β-conidia. (j) Culture from above on PDA medium. (k–m) Synnemata on the culture. (n) α-phialides. (o) β-phialides. (p) α-conidia. (q) β-conidia. Scale bars: (b, d, j) 1 cm, (c) 0.2 cm, (e) 300 μm, (f) 200 μm, (g, h) 10 μm, (i, p, q) 3 μm, (k, l) 0.5 cm, (m) 500 μm, (n, o) 20 μm.

Sexual morph: absent. Asexual morph: Synnemata generating from the fertile head of the host, single, light yellow, cylindrical, without a fertile head, stipitate, usually unbranched. Conidial mass yellowish, covered the surfaces of the host. α-phialides 9–15 × 1.1–2.2 μm (x¯ = 12 × 1.65 μm, n = 40), solitary, narrow lanceolate, from the synnema. β-phialides 19.8–25.9 × 1.7–2.6 μm (x¯ = 22.85 × 2.15 μm, n = 40), directly from hyphae, solitary, narrow lanceolate, suddenly tapering from the bottom to the apex. Conidia one-celled, hyaline, smooth, two types. α-conidia 2.1–2.5 μm (x¯ = 2.3 μm, n = 50), spherical, one-celled, smooth. β-conidia 2.9–3.8 × 1.3–2.2 μm (x¯ = 3.7 × 1.9 μm, n = 50), fusiform, one-celled, smooth.

Colonies on PDA medium slow-growing, isolated from the tissue of synnemata, circular, attaining 3 cm in 35 days at 25°C, dry yellow. Synnemata arising the margin of the colony after 30 days, without a fertile head, solitary or two- or three-branched, 2–6 × 0.9–1.8 mm (x¯ = 4 × 1.35 mm, n = 30), with several radiating ring-like distributions. Conidial masses pale yellow to yellow, covered the surface of the colony or generated from the middle part of the synnemata with hyaline to white yellow slime. Conidiophore 2–4 phialides in one. α-phialides 9–13.4 × 0.9–1.3 μm (x¯ = 11.2 × 1.1 μm, n = 40) unbranched, hyaline, smooth. β-phialides 12.8–20.9 × 1.9–2.8 μm (x¯ = 16.85 × 2.35 μm, n = 40), solitary, generating from hyphae laterally, hyaline, smooth. α-conidia 1.7–2.5 μm wide (x¯ = 2.1 μm, n = 50), globose, one-celled, smooth-walled; β-conidia 2.6–3.5 × 1.3–2.2 μm (x¯ = 3.05 × 1.75 μm, n = 50) hyaline, 1-celled, fusiform, smooth-walled.

Material examined: China, Anhui Province, Chuzhou City, parasitic on Paraisaria sp., on leaf litter, 25 August 2021, Yu Yang, HFS19a (GZLG 23-101, holotype; ex-type living culture, GZCC 22-2041).

Notes: Pleurocordyceps multisynnema has a high support value (100% ML/1 PP) and is sister to P. lanceolatus and P. marginaliradians in the phylogenetic tree (Figure 1). Comparing the ITS, LSU, SSU, tef-1α, rpb1, and rpb2 sequences of P. multisynnema and P. lanceolatus revealed 97.89% (12 bp differences), 99.28% (5 bp differences), 99.27% (6 bp differences), 99.77% (2 bp differences), 98.38% (11 bp differences), and 98.97% (10 bp differences) sequence similarities, respectively. Pleurocordyceps multisynnema differs from P. lanceolatus in that it is parasitic on Paraisaria species and produces conidia that range from coiled to thread-like but lack fertile heads (Xiao et al., 2023). Pleurocordyceps multisynnema differs from P. marginaliradians in distinct hosts (Paraisaria sp. vs. Cossidae larva), shorter phialides, and conidia (Xiao et al., 2018). As a result, Pleurocordyceps multisynnema is described as a new species of Pleurocordyceps.

Pleurocordyceps neoagarica Y. Yang and Y. P. Xiao sp. nov (Figure 4).

Index Fungorum number: IF900450; Faceoffungi number: FoF 14159

Etymology: Name referring to the similar species, Pleurocordyceps agarica.

Holotype: GZLG 23-103

Pleurocordyceps neoagarica (GZLG 23-103, Holotype). (a, b) Host: Ophiocordyceps neogryllotalpae (c) Upper side of the colony. (d, e) Synnemata on the culture. (f) Conidiophores. (g, h) Phialides. (i) Conidia. Scale bars: (b, c) 5 cm, (d) 3 mm, (e) 200 μm, (f) 20 μm, (g, h) 10 μm, (i) 2 μm.

Parasitism on Ophiocordyceps neogryllotalpae (Ophiocordycipitaceae, Hypocreales). Sexual morph: Not observed. Asexual morph: Hyphomycetous. Culture characteristics: Colonies on PDA quickly grown, isolated from the tissue, reaching 5 cm wide in 25 days at 25°C, white, reverse brown. Synnemata appearing after 15 days, 0.5–3 mm long, solitary, non-branched, displaying several ring-like distributions. Fertile head 1.2–2.3 mm wide, globose, pale yellow, producing from the top of the synnemata. Conidial masses covered the surface of synnemata or the top of synnemata, white yellow, slimy. Conidiophore 42–63 μm long (x¯ = 52.5 μm, n = 40), 2–4 phialides in one. Phialides 11.6–17.4 × 1.1–1.9 μm (x¯ = 14.5 × 1.5 μm, n = 50), one type, narrowly slim lanceolate, cylindrical at the base, 6–13 μm long, tapered into a long neck, 1.2–3.1 μm long, hyaline, smooth. Conidia 2.1–2.9 μm (x¯ = 2.5 μm, n = 50), arising from the apex of phialides, globose, 1-celled, hyaline.

Material examined: China, Guizhou Province, Qiannan Buyi and Miao Autonomous Prefecture, Sandu Shui Autonomous County. Parasitic on Ophiocordyceps neogryllotalpae (Ophiocordycipitaceae, Hypocreales), in the soil, 10 April 2022, Yu Yang, SD10H (GZLG 23-103, holotype; ex-type living culture, GZCC 22-2043).

Notes: Pleurocordyceps neoagarica (Host: Ophiocordyceps neogryllotalpae) differs from P. agarica (Host: Ophiocordyceps barnesii) morphologically due to its distinct host, longer synnemata and conidiophore, and shorter phialides (Wang et al., 2015b). P. neoagarica produces only one type of phialides and conidia, whereas P. agarica produces two. In the phylogenetic tree, the new collections (GZLG 23-103) shared a sister relationship with Pleurocordyceps agarica (Figure 1). The type of strain of P. neoagarica differs from P. agarica by 4 bp in ITS, 7 bp in SSU, 4 bp in rpb1, and 14 bp in rpb2 (Wang et al., 2015b). Given the significant morphological differences between these two taxa and their distinct phylogenetic placement, we conclude that they are separate species.

Pleurocordyceps sanduensis Y. P. Xiao and Y. Yang sp. nov (Figure 5).

Index Fungorum number: IF900447; Faceoffungi number: FoF 14157

Etymology: Name referring to the locality Sandu County.

Holotype: GZLG 23-104

Pleurocordyceps sanduensis (GZLG 23-104, Holotype). (a) Host: Ophiocordyceps neogryllotalpae (b, c) Upper and back side of the colony. (d, e) Conidial masses on the culture. (f) Conidiophores. (g) α-phialides. (h) β-phialides (I) α-conidia and β-conidia. Scale bars: (a) 5 cm; (b–d) 1 cm, (e) 0.5 cm, (f) 50 μm, (g) = 10 μm, (h) 20 μm, (i) 5 μm.

Parasite on Ophiocordyceps neogryllotalpae (Ophiocordycipitaceae, Hypocreales). Sexual morph: Not observed. Asexual morph: Hyphomycetous. Culture characteristics: Colonies on PDA fast-growing, obtained from tissue, reaching 5 cm wide in 20 days at 25°C, white, reverse yellow to brown, presenting multiple radiating ring-like distributions. Synnemata emerging after 25 days, solitary, unbranched, 0.1–0.5 mm long, distribution at the edge, with small or without a fertile head. Conidial masses covered the surface of the colony, pale yellow when young, later change to brown color, slime. Conidiophore 12–23 μm long (x¯ = 17.5 μm, n = 30), multiple phialides in one. Phialides exist in α-phialides and β-phialides. α-phialides 9.5–18.7 × 0.8–2.1 μm (x¯ = 14.1 × 1.45 μm, n = 40), smooth, hyaline, solitary. β-phialides 19–33.4 × 0.9–1.8 μm (x¯ = 26.2 × 1.35 μm, n = 40), smooth, hyaline, solitary. α-conidia 2.1–3.1 μm (x¯ = 2.6 μm, n = 50) wide, globose, unicellular, smooth-walled; β-conidia 3.3–5.5 × 1.5–2.1 μm (x¯ = 4.4 × 1.8 μm, n = 50) fusiform, unicellular, hyaline, smooth-walled.

Material examined: China, Guizhou Province, Qiannan Buyi and Miao Autonomous Prefecture, Sandu Shui Autonomous County. Parasitic on Ophiocordyceps neogryllotalpae associated with the larva of Gryllotalpa species, in soil, collected on 10 April 2022, Xingcan Peng, SD16 (GZLG 23-104, holotype; ex-type living culture, GZCC 22-2044).

Notes: Pleurocordyceps sanduensis (holotype: GZLG 23-104) is sister to P. clavisynnema (holotype: GZLG 23-102) with maximum statistical support (100% ML/1.00 PP) (Figure 1). Pleurocordyceps sanduensis is isolated from the same host as P. clavisynnema. However, the two are distinct in terms of both morphology and phylogeny. Base pair differences between P. clavisynnema and P. sanduensis are 23/824 in tef-1α, 8/1130 in SSU, 2/678 in rpb1, and 3/1050 in rpb2. Morphologically, Pleurocordyceps sanduensis differs from P. clavisynnema in shorter synnemata, smaller conidiophore, larger phialides, and longer conidia. Hence, this study introduces P. clavisynnema as a new species based on morphological and phylogenetic analyses.

 Discussion

Herein, we describe four new species of Pleurocordyceps (P. clavisynnema, P. multisynnema, P. neoagarica, and P. sanduensis) using a combination of morphology and phylogeny. The newly established species group distinctly form independent clades in the phylogenetic tree (Figure 1). Morphologically, three of the new species (P. clavisynnema, P. multisynnema, and P. sanduensis) are similar to P. aurantiacus, P. agarica, P. heilongtanensis, P. lanceolatus, P. marginaliradians, P. nutansis, P. sinensis, P. vitellina, and P. yunnanensis in that they have two types of phialides and conidia. However, the hosts on which P. clavisynnema, P. multisynnema, and P. sanduensis parasitize differ from those of other species of Pleurocordyceps (Wang et al., 2012, 2015a,b; Xiao et al., 2018, 2023). Meanwhile, P. neoagarica is similar to P. lianzhouensis and P. parvicapitata in that it has one type of phialides and conidia (Wang et al., 2014; Xiao et al., 2023). However, P. neoagarica differs from P. lianzhouensis and P. parvicapitata as it parasitizes different hosts and produces longer phialides and smaller conidia (Wang et al., 2014; Xiao et al., 2023).

The discovery of the new species of Pleurocordyceps adds to the diversity of the genus and the associated family. Several Pleurocordyceps taxa have been found in China, indicating a high diversity of these organisms in the country. Pleurocordyceps species display variable host specialization (Wang et al., 2012; Xiao et al., 2023). A few are host-specific. Herein, Pleurocordyceps clavisynnema, P. neoagarica, and P. sanduensis were isolated from the same host, Ophiocordyceps neogryllotalpae. This is similar to the previous results, whereby P. nutansis and P. yunnanensis are parasitic on the same fungus, Ophiocordyceps nutans (Wang et al., 2015a; Xiao et al., 2023). Most Pleurocordyceps taxa are not host-specific, and multiple species have been documented in the same host (Bischoff et al., 2003; Wang et al., 2012, 2015a,b; Matočec et al., 2014; Crous et al., 2017; Xiao et al., 2018). Members of the genus parasitize insects and fungi, several species of which have broad geographic distributions possibly reflecting the diversity of Pleurocordyceps habitats. Future studies should focus on collecting additional Polycephalomycetaceae taxa to not only uncover the full extent of diversity of this family but also understand their distribution in relation to their hosts.

 Data availability statement

The data presented in the study are deposited in the Guizhou Institute of Technology herbarium, accession number GZLG 23-102, GZCC 22-2042, GZLG 23-101, GZCC 22-2041, GZLG 23-103, GZCC 22-2043, GZLG 23-104, and GZCC 22-2044.

 Author contributions

Y-PX: Writing – original draft. YY: Writing – original draft. RJ: Writing – review & editing. EG: Writing – review & editing. X-CP: Formal analysis, Writing – review & editing. Z-LL: Writing – review & editing. Y-ZL: Writing – review & editing.

 Funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Guizhou Provincial Key Technology R&D Program [grant no. Qian Ke He Zhi Cheng (2021) Generally 200], Guizhou Province high-level talent innovation and entrepreneurship merit funding project (no. 202104), and Youth Science and Technology Talent Development Project from Guizhou Provincial Department of Education (QJHKYZ[2022]345).

YY would like to thank the Mushroom Research Foundation, Chiang Rai, Thailand for supporting this research. The authors also thank Dr. Shaun Pennycook (Landcare Research Manaaki Whenua, New Zealand) for advising on the fungal names.

 Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

 Publisher's note

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