11 urn:lsid:arphahub.com:pub:C004A564-9D6A-5F9F-B058-6A3815DFE9C3 urn:lsid:zoobank.org:pub:A4FD9303-3F01-45D9-B438-FCCFE465D2F7 MycoKeys MC 1314-4057 1314-4049 Pensoft Publishers 10.3897/mycokeys.105.119893 119893 Research Article Ascomycota Molecular systematics Taxonomy Asia Morphology and phylogeny of four new species within Polycephalomycetaceae (Hypocreales) parasitising Ophiocordyceps species Liu Zuoheng https://orcid.org/0000-0003-4118-3694 1 Tang Dexiang https://orcid.org/0000-0002-7662-224X 1 Lu Yingling https://orcid.org/0009-0008-8119-1975 1 Zhu Juye https://orcid.org/0000-0002-4184-5646 1 Luo Lijun https://orcid.org/0000-0002-1709-0781 1 Sun Tao https://orcid.org/0000-0001-7837-2101 1 Yu Hong hongyu@ynu.edu.cn https://orcid.org/0000-0002-2149-5714 1 Yunnan Herbal Laboratory, College of Ecology and Environmental Sciences, Yunnan University, Kunming, 650504, China Yunnan University Kunming China The International Joint Research Center for Sustainable Utilization of Cordyceps Bioresources in China and Southeast Asia, Yunnan University, Kunming, 650504, China Yunnan University Kunming China

Corresponding author: Hong Yu (hongyu@ynu.edu.cn,herbfish@163.com)

Academic editor: Marc Stadler

 2024 16 05 2024 105 179 202 4390F08B-4E8C-57E4-A2FA-50C997A9E606 31 01 2024 05 04 2024 Zuoheng Liu, Dexiang Tang, Yingling Lu, Juye Zhu, Lijun Luo, Tao Sun, Hong Yu This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Species of the family Polycephalomycetaceae grow on insects or entomopathogenic fungi and are distributed from tropical to subtropical regions. This study proposed four new species of hyperparasitic fungi from China based on six molecular markers (ITS, SSU, LSU, TEF-1α, RPB1 and RPB2) phylogenetic analyses and morphological characteristics. The four new species, i.e. Pleurocordyceps litangensis, Polycephalomyces jinghongensis, Po. multiperitheciatae and Po. myrmecophilus, were described and illustrated. Pl. litangensis, exhibiting a hyperparasitic lifestyle on Ophiocordyceps sinensis, differed from Pleurocordyceps other species in producing subulate β-phialides and ovoid or elliptic α-conidia. Po. jinghongensis was distinct from Polycephalomyces other species, being parasitic on Ophiocordyceps sp., as producing oval or long oval-shaped α-conidia and columns of β-conidia. Po. multiperitheciatae differed from Polycephalomyces other species as having synnemata with fertile head, linear β-conidia and parasitic on Ophiocordyceps multiperitheciata. Po. myrmecophilus was distinct from Polycephalomyces other species, being parasitic on the fungus Ophiocordyceps acroasca, as producing round or ovoid α-conidia and elliptical β-conidia without synnemata from the colonies. These four species were clearly distinguished from other species in the family Polycephalomycetaceae by phylogenetic and morphological characteristics. The morphological features were discussed and compared to relevant species in the present paper.

 entomogenous fungi hyperparasite micromorphology phylogenetic analyses taxonomy This work was supported by National Natural Science Foundation of China (No.31760011). Participation and sponsorship of the Yunnan University Professional Degree Graduate Practice Innovation Fund Program (ZC-22222937). Citation

Liu Z, Tang D, Lu Y, Zhu J, Luo L, Sun T, Yu H (2024) Morphology and phylogeny of four new species within Polycephalomycetaceae (Hypocreales) parasitising Ophiocordyceps species. MycoKeys 105: 179–202. https://doi.org/10.3897/mycokeys.105.119893

 Introduction

The new family Polycephalomycetaceae was established within clavicipitoid fungi to accommodate Perennicordyceps, Pleurocordyceps and Polycephalomyces based on morphology and phylogenetic analyses (Xiao et al. 2023). The genus Polycephalomyces Kobayasi was determined to be a monotypic anamorph genus for the species Polycephalomyces formosus Kobayasi (Kobayasi 1941). In the later taxonomic treatment of this genus, Seifert (1985) accepted four species, i.e. Po. formosus, Po. ramosus (Peck) Mains, Po. cylindrosporus Samson & H.C. Evans and Po. tomentosus (Schrader) Seifert. Polycephalomyces ditmarii Van Vooren & Audibert has been described as the asexual morph of Ophiocordyceps ditmarii (Quél.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora (Van Vooren and Audibert 2005). Paecilomyces sinensis C.T. Chen, S.R. Xiao & Z.Y. Shi was recombined into Polycephalomyces sinensis (Q.T. Chen, S.R. Xiao & Z.Y. Shi) W.J. Wang, X.L. Wang, Y. Li, S.R. Xiao & Y.J. Yao (Wang et al. 2012). The taxon has had a long history of being recognised as incertae sedis in Hypocreales (Kepler et al. 2013; Matočec et al. 2014). Matočec et al. (2014) established the genus Perennicordyceps and separated it from Polycephalomyces to accommodate Perennicordyceps cuboidea, Pe. paracuboidea, Pe. prolifica and Pe. ryogamiensis. Perennicordyceps was characterised by acremonium-like and hirsutella-like asexual morphs and perithecia (Xiao et al. 2023). Pleurocordyceps was established by combining the species originally assigned to the Polycephalomyces. Pl. sinensis was designated as the type species of the genus Pleurocordyceps (Wang et al. 2021).

Species of Polycephalomycetaceae grow on insects or other fungi, particularly Ophiocordyceps species and are distributed from tropical to subtropical regions (Bischof et al. 2003; Wang et al. 2012, 2015a, b; Matočec et al. 2014; Crous et al. 2017; Xiao et al. 2018; Poinar and Vega 2020). Several species of Polycephalomycetaceae have also been reported as hyperparasitic fungi, involving species of Cordyceps, Elaphomyces, Hirsutella, Myxomycetes and Ophiocordyceps (Seifert 1985; Bischof et al. 2003; Wang et al. 2012, 2015a, b).

South-western China is an area of high fungal biodiversity (Hyde et al. 2018). The rich biodiversity uncovered suggested that further collections could result in the discovery of numerous new taxa (Hyde et al. 2020a, b). In this study, the four novel species presented herein were collected from Yunnan Province and Sichuan Province in China. Morphological observations and phylogenetic analyses showed that these four species were novel and distinct from all other previously-described species in the family Polycephalomycetaceae. The four new species were discovered to be hyperparasites of Ophiocordyceps species. Pl. litangensis, Po. jinghongensis, Po. multiperitheciatae and Po. myrmecophilus were hyperparasitic on O. sinensis, Ophiocordyceps sp., O. multiperitheciata and O. acroasca, respectively. At present, relatively little is known about the mechanisms responsible for hyperparasitism in species of the family Polycephalomycetaceae and our findings provide ideal material for these studies. These findings have expanded the diversity of fungal species in the family Polycephalomycetaceae, providing taxonomic data to support species resource conservation and rational exploitation and utilisation of resources.

 Materials and methods Specimens and isolates

Fungal specimens parasitising Ophiocordyceps sp. were collected from different regions of south-western China, including Sichuan Province (Litang County) and Yunnan Province (Jinghong City, Yuanyang County, Pu'er City). The specimens were found in moist soils. Geographic information (longitude, latitude and altitude) of collection were recorded in the field, then specimens were collected in sterilised plastic containers and transported to the laboratory. The micro-morphological characters (Synnemata) were examined using an Olympus SZ61 stereomicroscope (Olympus Corporation, Tokyo, Japan). To obtain axenic culture, the stromata was divided into 2–4 segments with sterilised blades. Each segment was immersed in hydrogen peroxide 30% (H2O2) for 5 min and then rinsed five times in sterile water. After drying on sterilised filter paper, these segments were inoculated on Potato Dextrose Agar (PDA) plates. The conidial masses at the apex of the stipes were picked with an inoculating needle and immersed in 5 ml of sterilised water for blending. The homogenates were then spread on PDA plates containing 0.1 g/l streptomycin and 0.05 g/l tetracycline. The plates were maintained in a culture room at 25 °C. After purification, the cultures were stored at 4 °C (Wang et al. 2015a). Dry specimens were deposited in the Yunnan Herbal Herbarium (YHH) of Yunnan University. The cultures were stored in Yunnan Fungal Culture Collection (YFCC) of Yunnan University.

 Morphological studies

Cultures on potato extract agar (PDA) were incubated for 21 days at 25 °C and photographed using a Canon 750 D camera (Canon Inc., Tokyo, Japan). For asexual morphological descriptions, microscope slide cultures were prepared by placing a small amount of mycelium on 5 mm diameter PDA medium blocks that were overlaid by a cover slip (Wang et al. 2015a; Tang et al. 2023b). The observations, measurements and photographs of the phialides and conidia were made using a light microscope (Olympus BX53).

 DNA extraction, <abbrev xlink:title="polymerase chain reaction" id="ABBRID0E3HAE">PCR</abbrev> and sequencing

DNA templates were obtained from cultures using the CTAB method, following that described in Liu et al. (2001). The polymerase chain reaction (PCR) was used to amplify genetic markers using the following primer pairs: ITS4/ITS5 for ITS (internal transcribed spacer gene region) (White et al. 1990), NS1/NS4 for SSU (small subunit ribosomal RNA gene region) (White et al. 1990), LR0R/LR5 for LSU (large subunit rRNA gene region) (Hopple 1994) 2218R/983F for TEF-1α (translation elongation factor 1-alpha gene region) (Rehner and Buckley 2005), CRPB1/RPB1Croph for RPB1 (RNA polymerase II largest subunit gene region) (Castlebury et al. 2004; Araújo et al. 2018), fRPB2-7cR/fRPB2-5F for RPB2 (RNA polymerase II second largest subunit) (Liu et al. 1999). A total of 25 µl PCR matrix contained PCR 2.5 µl Buffer (Transgen Biotech, Beijing, China), 17.25 µl sterile water, 4 µl dNTP, 1 µl each forward and reverse primer, 0.25 µl Taq DNA polymerase (Transgen Biotech, Beijing, China) and 1 µl DNA template. The matrix and reactions conditions were prepared and performed according to the methods described in previous studies (Xiao et al. 2023).

 Phylogenetic analysis

In order to construct a phylogeny of the major lineages in the family Polycephalomycetaceae, most of the DNA sequences used in this work were derived from previous phylogenetic studies (Xiao et al. 2023). Phylogenetic analyses were based on sequences of six molecular markers (ITS, SSU, LSU, TEF-1α, RPB1 and RPB2), all of which were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/). Then the nucleotide sequences were combined with those generated in our study (Table 1). Sequences were aligned using ClustalX v.2.0 (Larkin et al. 2007), adjusted manually and then concatenated in BioEdit v.7.1.1 (Hall 1999). Poorly-aligned regions were removed and adjusted manually using MEGA6 (v.6.0) (Tamura et al. 2013). ModelFinder (Kalyaanamoorthy et al. 2017) was used to select the best-fitting likelihood model for the Maximum likelihood (ML) analyses and the Bayesian inference (BI) analyses were carried out for the fungi datasets. For ML analyses, tree searches were performed in IQ-tree (v.2.1.3) (Nguyen et al. 2015), based on the best-fit model GTR+F+I+I+R3 with 5000 ultrafast bootstraps (Hoang et al. 2017) in a single run. The BI search was according to the best-fit model GTR+F+I+G4, resorting to MrBayes (v.3.2.2) for BI analysis (Ronquist et al. 2012). The phylogenetic trees constructed using the ML and the BI analyses were largely congruent and strongly supported in most branches (Fig. 1). The final phylogenetic tree was visualised with its Maximum-Likelihood bootstrap proportions (ML-BS) and Bayesian posterior probability (BI-BPP) performed using FigTree v.1.4.2 and edited via Adobe Illustrator CS6.

10.3897/mycokeys.105.119893.figure1 4D43D3C2-551E-5983-BDE6-3F6401FE41A9

Phylogenetic tree of Polycephalomycetaceae, based on the concatenation of ITS, SSU, LSU, TEF-1α, RPB1 and RPB2 sequence data. The tree was generated from an alignment of 6,384 sites and 113 taxa. The phylogeny was inferred using the IQ-tree. The Maximum likelihood bootstrap values greater than 75% (on the left) and the Bayesian posterior probabilities over 0.75 (on the right) were indicated above the nodes. The new species were indicated in back bold font.

https://binary.pensoft.net/fig/1050580

Sources of selected isolates and GenBank accession number for ITS and five genes of three genera within Polycephalomycetaceae were used in this study.

Species name Voucher ITS SSU LSU TEF-1α RPB1 RPB2 References
Cordyceps pleuricapitata NBRC 109979 AB925941 AB925978 Unpublished
Cordyceps pleuricapitata NBRC 109978 AB925940 AB925977 Unpublished
Cordyceps pleuricapitata NBRC 109977 AB925939 AB925976 Unpublished
Cordyceps pleuricapitata NBRC 100746 JN943306 JN941749 JN941392 KF049680 JN992483 KF049668 Kepler et al. (2013)
Cordyceps pleuricapitata NBRC 100745 JN943304 JN941750 JN941391 KF049679 Kepler et al. (2013)
Perennicordyceps elaphomyceticola MFLU 21-0262 OQ172064 OQ172101 OQ172032 OQ459718 OQ459747 OQ459792 Xiao et al. (2023)
Perennicordyceps cuboidea NBRC 100941 JN943329 JN941725 JN941416 JN992459 Schoch et al. (2012)
Perennicordyceps cuboidea NBRC 103834 JN943330 JN941723 JN941418 JN992457 Schoch et al. (2012)
Perennicordyceps cuboidea NBRC 103835 JN943333 JN941722 JN941419 JN992456 Schoch et al. (2012)
Perennicordyceps elaphomyceticola MFLU 21-0264 OQ172067 OQ172103 OQ172035 OQ459720 OQ459749 OQ459794 Xiao et al. (2023)
Perennicordyceps elaphomyceticola MFLU 21-0266 OQ172068 OQ172112 OQ172036 OQ459732 OQ459760 OQ459806 Xiao et al. (2023)
Perennicordyceps elaphomyceticola MFLU 21-0263 OQ172065 OQ172102 OQ172033 OQ459719 OQ459748 OQ459793 Xiao et al. (2023)
Perennicordyceps elaphomyceticola NTUCC 17-022 MK840824 MK840813 MK839230 MK839221 MK839212 Yang et al. (2020)
Perennicordyceps lutea KUMCC 3004 OQ474910 Xiao et al. (2023)
Perennicordyceps paracuboidea NBRC 100942 JN943337 JN941711 JN941430 JN992445 AB972958 Schoch et al. (2012)
Perennicordyceps prolifica NBRC 103839 JN943342 JN941706 JN941435 JN992440 Schoch et al. (2012)
Perennicordyceps prolifica NBRC 103838 JN943339 JN941707 JN941434 JN992441 Schoch et al. (2012)
Perennicordyceps prolifica TNS-F-18547 KF049660 KF049613 KF049632 KF049687 KF049649 KF049670 Kepler et al. (2013)
Perennicordyceps prolifica TNS-F-18481 KF049659 KF049612 KF049631 KF049686 KF049648 Kepler et al. (2013)
Perennicordyceps ryogamiensis NBRC 101751 JN943343 JN941703 JN941438 KF049688 JN992437 Schoch et al. (2012)
Perennicordyceps ryogamiensis NBRC 103837 JN943346 JN941702 JN941439 JN992436 Schoch et al. (2012)
Perennicordyceps ryogamiensis NBRC 103842 JN943345 JN941701 JN941440 JN992435 Schoch et al. (2012)
Pleurocordyceps parvicapitata MFLU 21-0270 OQ172082 OQ172105 OQ172054 OQ459722 OQ459751 OQ459796 Xiao et al. (2023)
Pleurocordyceps agarica YHHPA 1305T KP276651 KP276655 KP276659 KP276663 KP276667 Wang et al. (2015b)
Pleurocordyceps agarica YHCPA1307 KP276654 KP276658 KP276662 KP276666 KP276670 Wang et al. (2015b)
Pleurocordyceps agarica YHCPA 1303 KP276653 KP276657 KP276661 KP276665 KP276669 Wang et al. (2015b)
Pleurocordyceps aurantiaca MFLUCC 17-2113T MG136916 MG136904 MG136910 MG136875 MG136866 MG136870 Xiao et al. (2019)
Pleurocordyceps aurantiaca MFLUCC 17-2114 MG136917 MG136905 MG136911 MG136874 MG136871 Xiao et al. (2019)
Pleurocordyceps aurantiaca MFLU 17-1394 MG136918 MG136906 MG136912 MG136876 MG136867 MG136872 Xiao et al. (2019)
Pleurocordyceps aurantiaca MFLU 17-1393T MG136907 MG136913 MG136877 MG136868 MG136873 Xiao et al. (2019)
Pleurocordyceps ramosus like NBRC 101760 MN586827 MN586818 MN586836 MN598051 MN598042 MN598060 Wang et al. (2020)
Pleurocordyceps ramosus like NBRC 109984 MN586828 MN586819 MN586837 MN598052 MN598043 Wang et al. (2020)
Pleurocordyceps ramosus like NBRC 109985 MN586829 MN586820 MN586838 MN598053 MN598044 Wang et al. (2020)
Pleurocordyceps heilongtanensis KUMCC 3008 OQ172091 OQ172111 OQ172063 OQ459731 OQ459759 OQ459805 Xiao et al. (2023)
Pleurocordyceps kanzashianus AB027371 AB027325 AB027371 Nikoh et al. (2000)
Pleurocordyceps lanceolatus GACP 17-2004T OQ172076 OQ172110 OQ172046 OQ459726 OQ459754 OQ459800 Xiao et al. (2023)
Pleurocordyceps lanceolatus GACP 17-2005T OQ172109 OQ172047 OQ459727 OQ459755 OQ459801 Xiao et al. (2023)
Pleurocordyceps lianzhouensis HIMGD20918T EU149921 KF226245 KF226246 KF226248 KF226247 Zhang et al. (2007)
Pleurocordyceps lianzhouensis GIMYY9603 EU149922 KF226249 KF226250 KF226252 KF226251 Zhang et al. (2007)
Pleurocordyceps marginaliradians MFLU 17-1582T MG136920 MG136908 MG136914 MG136878 MG136869 MG271931 Xiao et al. (2019)
Pleurocordyceps marginaliradians MFLUCC 17-2276T MG136921 MG136909 MG136915 MG136879 MG271930 Xiao et al. (2019)
Pleurocordyceps nipponica BCC 1682 KF049664 KF049620 KF049638 KF049694 Kepler et al. (2013)
Pleurocordyceps nipponica BCC 18108 KF049657 MF416624 MF416569 MF416517 MF416676 MF416462 Kepler et al. (2013)
Pleurocordyceps nipponica NBRC 101407 JN943302 JN941752 JN941389 JN992486 Schoch et al. (2012)
Pleurocordyceps nipponica NBRC 101405 JN943442 JN941754 JN941387 JN992488 Schoch et al. (2012)
Pleurocordyceps nipponica BCC 2325 KF049665 KF049622 KF049640 KF049696 KF049655 KF049677 Kepler et al. (2013)
Pleurocordyceps nipponica NHJ 4268 KF049621 KF049639 KF049695 KF049654 KF049676 Kepler et al. (2013)
Pleurocordyceps nipponica BCC 1881 KF049618 KF049636 KF049692 KF049674 Kepler et al. (2013)
Pleurocordyceps nutansis GACP 19-1906 OQ172079 OQ172117 OQ172049 OQ459737 OQ459763 OQ459809 Xiao et al. (2023)
Pleurocordyceps nutansis GACP 19-1907 OQ172087 OQ172118 OQ172059 OQ459738 OQ459764 OQ459810 Xiao et al. (2023)
Pleurocordyceps nutansis GACP 19-3019T OQ172086 OQ172120 OQ172058 OQ459740 OQ459766 OQ459812 Xiao et al. (2023)
Pleurocordyceps nutansis MFLU 21-0275T OQ172073 OQ172119 OQ172048 OQ459739 OQ459765 OQ459811 Xiao et al. (2023)
Pleurocordyceps onorei BRA CR23904 KU898843 Crous et al. (2017)
Pleurocordyceps onorei BRA CR23902T KU898841 Crous et al. (2017)
Pleurocordyceps ophiocordycipiticola MFLUCC 22-0187 NR185465 NG229093 Wei et al. (2022)
Pleurocordyceps ophiocordycipiticola MFLU:22-0265 OQ127364 OQ127326 OQ127397 OQ186388 OQ186435 Wei et al. (2022)
Pleurocordyceps parvicapitata MFLU 21-0271T OQ172083 OQ172106 OQ172055 OQ459723 OQ459752 OQ459797 Xiao et al. (2019)
Pleurocordyceps parvicapitata MFLU 21-0272 OQ172084 OQ172099 OQ172056 OQ459716 OQ459745 OQ459790 Xiao et al. (2023)
Pleurocordyceps parvicapitata MFLU 21-0273 OQ172085 OQ172100 OQ172057 OQ459717 OQ459746 OQ459791 Xiao et al. (2023)
Pleurocordyceps phaothaiensis BCC84553T MF959733 MF959737 MF959742 MF959745 Crous et al. (2017)
Pleurocordyceps phaothaiensis BCC84552 MF959732 MF959736 MF959740 MF959744 Crous et al. (2017)
Pleurocordyceps phaothaiensis BCC84551 MF959731 MF959735 MF959739 MF959743 Crous et al. (2017)
Pleurocordyceps ramosopulvinata EFCC 5566 KF049627 KF049682 KF049645 Kepler et al. (2013)
Pleurocordyceps ramosopulvinata SU 65 DQ118742 DQ118753 DQ127244 Chaverri et al. (2005)
Pleurocordyceps sinensis ARSEF 1424 KF049661 KF049615 KF049634 KF049689 KF049671 Kepler et al. (2013)
Pleurocordyceps sinensis CN 80-2T HQ832884 HQ832887 HQ832886 HQ832890 HQ832888 HQ832889 Wang et al. (2012)
Pleurocordyceps sinensis HMAS 43720T NR119928 NG042573 Wang et al. (2012)
Pleurocordyceps sinensis MFLU 21-0269 OQ172080 OQ172122 OQ172050 OQ459742 OQ459768 Xiao et al. (2023)
Pleurocordyceps sinensis GACP 20-2305 OQ172075 OQ172108 OQ172045 OQ459725 OQ459753 OQ459799 Xiao et al. (2023)
Pleurocordyceps sinensis GACP 20-2304 OQ172074 OQ172107 OQ172044 OQ459724 OQ459798 Xiao et al. (2023)
Pleurocordyceps sinensis GZU 20-0865 OQ172071 OQ172096 OQ172043 OQ459713 Xiao et al. (2023)
Pleurocordyceps sinensis MFLU 21-0268 OQ172070 OQ172123 OQ172052 OQ459743 OQ459815 Xiao et al. (2023)
Pleurocordyceps sinensis MFLU 21-0267 OQ172121 OQ172051 Xiao et al. (2023)
Pleurocordyceps sinensis MFLU 18-0162 MK863250 MK863043 MK863050 MK860188 Unpublished
Pleurocordyceps sp. BCC 2637 KF049663 KF049637 KF049693 KF049675 Kepler et al. (2013)
Pleurocordyceps sp. JB07.08. 16_8 KF049662 KF049616 KF049635 KF049690 KF049652 KF049672 Kepler et al. (2013)
Pleurocordyceps sp. JB07.08.17_07b KF049617 KF049691 KF049653 KF049673 Kepler et al. (2013)
Pleurocordyceps sp. NBRC 109987 AB925983 Unpublished
Pleurocordyceps sp. NBRC 109988 AB925984 Unpublished
Pleurocordyceps sp. NBRC 109990 AB925968 Unpublished
Pleurocordyceps sp. NBRC 110224 AB925969 Unpublished
Pleurocordyceps litangensis YFCC 06109293 PP410597 PP541902 PP410593 PP550103 PP697751 This study
Pleurocordyceps litangensis YFCC 06109294 PP410598 PP541903 PP410594 PP550104 PP697752 PP550107 This study
Pleurocordyceps litangensis YFCC 06109295 PP410600 PP541905 PP410596 PP550106 PP697754 This study
Pleurocordyceps litangensis YFCC 06109296 PP410599 PP541904 PP410595 PP550105 PP697753 PP550108 This study
Pleurocordyceps sp. GIMCC 3.570 JX006097 JX006098 JX006100 JX006101 Zhong et al. (2016)
Pleurocordyceps tomentosus BL4 KF049666 KF049623 KF049641 KF049697 KF049656 KF049678 Kepler et al. (2013)
Pleurocordyceps vitellina KUMCC 3005 OQ172088 OQ172060 OQ459728 OQ459756 OQ459802 Xiao et al. (2023)
Pleurocordyceps vitellina KUMCC 3006 OQ172089 OQ172061 OQ459729 OQ459757 OQ459803 Xiao et al. (2023)
Pleurocordyceps vitellina KUMCC 3007 OQ172090 OQ172062 OQ459730 OQ459758 OQ459804 Xiao et al. (2023)
Pleurocordyceps yunnanensis YHCPY1005 KF977848 KF977850 KF977852 KF977854 Wang et al. (2015a)
Pleurocordyceps yunnanensis YHHPY1006T KF977849 KF977851 KF977853 KF977855 Wang et al. (2015a)
Polycephalomyces albiramus GACP 21-XS08T OQ172092 OQ172115 OQ172037 OQ459735 OQ459761 OQ459807 Xiao et al. (2023)
Polycephalomyces albiramus GACPCC 21-XS08T OQ172093 OQ172116 OQ172038 OQ459736 OQ459762 OQ459808 Xiao et al. (2023)
Polycephalomyces formosus NBRC 109993T MN586833 MN586824 MN586842 MN598057 MN598048 MN598064 Wang et al. (2021)
Polycephalomyces formosus NBRC 109994 MN586834 MN586825 MN586843 MN598058 MN598049 MN598065 Wang et al. (2021)
Polycephalomyces formosus NBRC 109995 MN586835 MN586826 MN586844 MN598059 MN598050 MN598066 Wang et al. (2021)
Polycephalomyces formosus GACP 21-WFKQ04 OQ172095 OQ172114 OQ172040 OQ459734 Xiao et al. (2023)
Polycephalomyces jinghongensis YFCC 02959283 PP274089 PP274093 PP274109 PP581803 PP697747 PP581819 This study
Polycephalomyces jinghongensis YFCC 02959284 PP274090 PP274094 PP274110 PP581804 PP697748 PP581820 This study
Polycephalomyces jinghongensis YFCC 02959285 PP274091 PP274095 PP274111 PP581805 PP697749 PP581821 This study
Polycephalomyces jinghongensis YFCC 02959286 PP274092 PP274096 PP274112 PP581806 PP697750 PP581822 This study
Polycephalomyces multiperitheciatae YFCC 06149287 PP274102 PP274108 PP274118 PP581802 PP581818 This study
Polycephalomyces multiperitheciatae YFCC 06149288 PP274098 PP274104 PP274114 PP581798 PP697743 PP581815 This study
Polycephalomyces multiperitheciatae YFCC 06149289 PP274101 PP274107 PP274117 PP581801 PP697746 PP581817 This study
Polycephalomyces multiperitheciatae YFCC 06149290 PP274097 PP274103 PP274113 PP581797 PP697742 PP581814 This study
Polycephalomyces multiperitheciatae YFCC 06149291 PP274100 PP274106 PP274116 PP581800 PP697745 This study
Polycephalomyces multiperitheciatae YFCC 06149292 PP274099 PP274105 PP274115 PP581799 PP697744 PP581816 This study
Polycephalomyces myrmecophilus YFCC 09289443 PP410602 PP410608 PP410605 PP581795 PP697740 PP581812 This study
Polycephalomyces myrmecophilus YFCC 09289444 PP410603 PP410609 PP410606 PP581796 PP697741 PP581813 This study
Polycephalomyces myrmecophilus YFCC 09289445 PP410601 PP410607 PP410604 PP581794 PP697739 PP581811 This study
Tolypocladium ophioglossoides NBRC 100998 JN943319 JN941735 JN941406 AB968602 JN992469 AB968563 Ban et al. (2015)
Tolypocladium ophioglossoides NBRC 106330 JN943321 JN941734 JN941407 AB968603 JN992468 AB968564 Ban et al. (2015)
Results Phylogenetic tree

Sequences of 113 samples were used for phylogenetic analysis. Tolypocladium ophioglossoides (NBRC 106330) and T. ophioglossoides (NBRC 100998) were designated as the outgroup taxa (Xiao et al. 2023). The total length of the concatenated dataset of six genes across the 113 samples was 6384 bp, including 859 bp for ITS, 1548 bp for SSU, 930 bp for LSU, 1037 bp for TEF-1α, 797 bp for RPB1 and 1213 bp for RPB2. The phylogenetic relationships show three major clades within the family Polycephalomycetaceae (Fig. 1), consisting of the clade Pleurocordyceps (16 species; BS = 100%, BPP = 1.00), the clade Perennicordyceps (6 species; BS = 100%, BPP = 1.00) and the clade Polycephalomyces (6 species; BS = 100%, BPP = 1.00). Pleurocordyceps nutansis, Pleurocordyceps sinensis (MFLU 21-0268, GZU 20-0865) are adjacent clades. Similarly, Pleurocordyceps ramosus like and Pleurocordyceps yunnanensis are contiguous branches. In addition, Pleurocordyceps kanzashianus is included in the clade Pleurocordyceps nipponica. Cordyceps pleuricapitata strains also formed a monophyletic clade (BS = 100%, BPP = 1.00). The four species collected and described in this work are clustered in the clade Pleurocordyceps (Pl. litangensis) and the clade Polycephalomyces (Po. jinghongensis, Po. multiperitheciatae and Po. myrmecophilus), respectively.

 Taxonomy Fungi Hypocreales Ophiocordycipitaceae 982CA96F-8B9B-5C9F-8EC0-C88D811E3621 Pleurocordyceps litangensis 851497 Hong Yu bis, Z.H. Liu & D.X. Tang sp. nov. Fig. 2 Etymology.

litangensis = Litang County, the epithet referred to the nature study trail in Litang County, the locality where the type specimen was collected.

 Diagnosis.

Pleurocordyceps litangensis and Pl. sinensis have the same host (O. sinensis) and β-Conidia, but the phialides (lanceolate or narrowly lageniform vs. spear point or subulate), α-conidia (Ovoid vs. Ovoid or ellipticare) are different.

10.3897/mycokeys.105.119893.figure2 5D6A14DE-F5BC-568A-842A-E497D34348E4

Morphological features of Pleurocordyceps litangensis (Holotype: YHH 2306055) a overview of Pleurocordyceps litangensis and its host b synnemata on the insects c, d colony obverse and reverse e–h, k α-phialides i α-conidia j β-conidia and β-phialides. Scale Bars: 2 cm (a–d); 20 μm (e–j); 10 μm (k); 5 μm (g–i).

https://binary.pensoft.net/fig/1050581 Holotype.

China, Sichuan Province, Ganzi Tibetan Autonomous Prefecture, Litang County, parasitic on Ophiocordyceps sinensis (Ophiocordycipitaceae), on insects buried in soil, with erect stromata, 30°43′00″N, 100°52′00″E, alt. 4750 m, 10 June 2023, Hong Yu bis (YHH 2306055).

 Sexual morph.

Undetermined.

 Asexual morph.

Synnemata arising from the stromata of O. sinensis, solitary or alternating; clavate or spatulate, branched and unbranched, straight or sinuous. Terminal portion of a synnemata covered by a viscous mass, khaki. Colonies on PDA growing slowly, attaining a diameter of 1.4–1.6 cm in 3 weeks at 25 °C, filiform, dark yellow and reverse dry yellow. Phialides existing in two types: α- and β-phialides. Both types of phialides often reproduce new phialides at their own apices and yield catenulate β-conidia, collarettes not flared, periclinal thickening not visible. α-phialides acropleurogenous solitary on hyphae; spear point, tapering gradually from the base to the apex, 11.2–12.8 μm long, 1.9–2.6 μm wide at the base and 0.7–0.9 μm wide at the apex. β-phialides terminal on solitary on hyphae; subulate, tapering abruptly from the base to the apex, 9.9–27.8 μm long, 1.6–2.5 μm wide at the base and 0.6–1.4 μm wide at the apex. α-conidia ovoid or elliptic and occurring on the final portion of synnemata, 3.2–6.1 × 1.8–3.9 μm; β-conidia fusiform, and produce on the surface mycelium of colony, multiple, usually in chains on a phialide, 3.5–6.1 × 1.4–2.5 μm.

 Host.

Parasitic on Ophiocordyceps sinensis (Ophiocordycipitaceae).

 Distribution.

China, Sichuan Province.

 Material examined.

China, Sichuan Province, Ganzi Tibetan Autonomous Prefecture, Litang County, parasitic on Ophiocordyceps sinensis (Ophiocordycipitaceae), on insects buried in soil, with erect stromata, 30°43′00″N, 100°52′00″E, alt. 4750 m, 10 June 2023, Tao Sun. Paratypes: YHH 2306058; other collections: YHH 2306059; Culture ex-type: YFCC 06109293; Other living cultures: YFCC 06109294, YFCC 06109295, YFCC 06109296.

 Notes.

Four strains, Pleurocordyceps sp. NBRC109990, Pl. sp. NBRC109987, Pl. sp. NBRC110224, Pl. sp. NBRC109988, were aggregated Pl. litangensis into one branch (Fig. 1 BS = 100%, BPP = 1.00). Pl. litangensis was distinct from other species of Pleurocordyceps by α-phialides spear point, β-phialides subulate, α-conidia ovoid or elliptic (Table 2). Thus, Pl. litangensis was introduced as a new species under the genus Pleurocordyceps.

Morphological comparison of asexual morph species of Pleurocordyceps.

Species Host Synnemata Phialides Conidia References
Pl. agarica Ophiocordyceps sp. or melolonthid larvae Solitary, unbranched, agaricshaped; conidial mass pileus-like, light yellow to pale brown α-phialides lanceolate; β-phialides narrowly lageniform or subulate α-conidia globose to subglobose; β-conidia fusiform, catenate or clump together Wang et al. (2015b)
Pl. aurantiacus Coleoptera larvae or O. barnesii Emerging after 30 days, solitary or not solitary, branched or unbranched, showing 1–2 radiating ring like distributions α-phialides, narrowly lageniform. β-phialides, lanceolate or narrowly lageniform α-conidia, globose to subglobose. β-conidia, fusiform Xiao et al. (2018)
Pl. lanceolatus Lepidoptera larvae Lanceolate to corniform, solitary to crowded, stipitate, usually unbranched, rarely branched on the PDA, yellow to yellowish on the fresh specimen, covered with conidial masses, white on the PDA α-phialides directly from hyphae, solitary, usually unbranched, subulate, at the base, tapering into a long neck; β-phialides branched into 2 or 3 phial ides, narrowly lageniform to lanceolate α-conidia spherical, forming slimy conidial masses along the Synnemata; β-conidia fusiform Xiao et al. (2023)
Pl. marginaliradians Cossidae larva Emerging after 14 days, single or branched into 2 or 3 branched, showing 1–2 radiating ring like distributions α-phialides, elongate lageniform; β-phialides, narrow slender to narrow lageniform α-conidia globose, catenate, one-celled, pale yellow slimy in mass. β-conidia fusiform, one-celled Xiao et al. (2018)
Pl. parvicapitata Perennicordyceps elaphomyceticola Absent Phialides, cylindrical at the base, tapering into a long neck globose to subglobose Xiao et al. (2023)
Pl. sinensis Lepidoptera larvae or Ophiocordyceps sinensis Solitary, crowded, branched or unbranched, conidial mass yellow or yellow-orange Lanceolate or narrowly lageniform α-conidia, ovoid; β-conidia, fusiform Chen et al. (1984); Wang et al. (2012)
Pl. vitellina Ophiocordyceps nigrella Absent α-phialides, hyaline, smooth, elongated lageniform, crowed, gathered in the middle of colony. β-phialides, hyaline, smooth, directly growing from hyphae, with or without metula at the base, solitary, lanceolate, ovate at the base, tapering into a short neck α-conidia spherical, one-celled, smooth-walled. β-conidia fusiform, catenulate Xiao et al. (2023)
Pl. yunnanensis Hemiptera adults or Ophiocordyceps nutans Solitary, caespitose or crowded, branched or unbranched; conidial mass white to yellow–brown α-phialides cylindrical to subulate; β-phialides narrowly lageniform or subulate α-conidia subglobose, ellipsoidal; β-conidia fusiform, catenate or clump together Wang et al. (2015a)
Pl. nutansis Ophiocordyceps nutans Cylindrical, clavate, capitate, stipitate, crowded, simple, white to yellowish Two types, both of the types observed on the same synnemata. α-phialides, gathered at the apex of the synnemata, arranged in a parallel palisade-like layer around the apex of the fertile head, hyaline, usually branched into 2–6 phialides, narrowly slender lanceolate; β-phialides , solitary, scattered along the stipe, lanceolate, ovate at the base, tapering into a long neck α-conidia, spherical, forming slimy conidial masses on the fertile head; β-conidia fusiform, produced along stipe of the synnemata Xiao et al. (2023)
Pl. heilongtanensis Ophiocordyceps sp. Scattered on the surface of host, cylindrical, stipitate, unbranched, white, with or without fertile head α-phialides, hyaline, smooth, elongated lageniform, caespitose, palisade-like, crowed, gathered in the top of synnemata, mostly branched into 2–4 phialides. β-phialides hyaline, smooth, solitary, branched into 2 or 3 phial ides, with or without metula at the base, directly growing from hyphae α-conidia, subglobose to ovoid,in yellowish slimy mass. β-conidia fusiform, one-celled Xiao et al. (2023)
Pl. lianzhouensis Lepidoptera larva or Ophiocordyceps crinalis Unbranched or dichotomously branched, conidial mass not seen In whorls or intercalary and terminal, terminally awl-shaped Ellipsoidal, oblong to cylindrical Wang et al. (2014)
Pl. litangensis Ophiocordyceps sinensis Absent α-phialides acropleurogenous solitary on hyphae; spear point. β-phialides terminal on solitary on hyphae; subulate α-conidia ovoid or elliptical; β-conidia fusiform This study
Fungi Hypocreales Ophiocordycipitaceae 0FA1388C-4371-5409-8EBA-A9F4CABEE4D1 Polycephalomyces jinghongensis 851498 Hong Yu bis, Z.H. Liu & D.X. Tang sp. nov. Fig. 3 Etymology.

jinghongensis = Jinghong City, the epithet referred to the nature study trail in Jinghong City, the locality where the type specimen was collected.

 Diagnosis.

Polycephalomyces jinghongensis are similar to that of Po. multiperitheciatae regarding the production of α-conidia oval, but Po. jinghongensis differ by synnemata caespitose, white to orange-yellow colour, producing cylindrical β-conidia, parasitic on Ophiocordyceps sp.

 Holotype.

China, Yunnan Province, Xishuangbanna Dai Autonomous Prefecture, Jinghong City, parasitic on Ophiocordyceps sp. (Ophiocordycipitaceae), on insects buried in soil, with erect synnemata, 23°47′9″N, 102°51′41″E, alt. 2053 m, 25 September 2022, Hong Yu bis (YHH 2206047).

 Sexual morph.

Undetermined.

 Asexual morph.

Synnemata arising from the stromata of Ophiocordyceps sp., 0.8–1.6 cm long 0.1–0.3 cm thick, caespitose, unbranched or branched, white to orange-yellow colour. Colonies on PDA growing slowly, attaining a diameter of 1.3–1.7 cm in 3 weeks at 25 °C, clustered, white and reverse dry yellow. Synnemata emerging after 14 days, tufted, branched and 0.6–10 mm long, showing radiating distributions. Phialides existing in two types: α- and β-phialides. Both types of phialides often reproduce new phialides at their own apices or sides, collarettes not flared, periclinal thickening not visible. α-phialides verticillate and acropleurogenous on conidiophores and solitary on hyphae; lanceolate, tapering gradually from the base to the apex, 4.5–19.5 μm long, 1.4–2.5 μm wide at the base and 0.8–1.6 μm wide at the apex. β-phialides acropleurogenous in whorls of 2–3 or intercalary and terminal on conidiophores and solitary on hyphae; diamond-shaped; tapering abruptly from the base to the apex, 10.4–17.5 μm long, 1.1–2.7 μm wide at the base, and 0.4–1.1 μm wide at the apex. α-conidia oval or long oval shape and occurring in the conidial mass on the agar or on the final portion of synnemata, 1.1–3.4 × 0.8–1.9 μm; β-conidia columns and produced on the surface mycelium of colony, multiple, usually formed as spore balls at the phialidic apex, 2.3–3.1 × 1.2–1.3 μm.

10.3897/mycokeys.105.119893.figure3 9D15822B-7A72-50DE-B6A0-DCCF3BD42730

Morphological features of Polycephalomyces jinghongensis (Holotype: YHH 2209031) a overview of Polycephalomyces jinghongensis and its host b synnemata on the insect c, d colony obverse and reverse e–g β-phialides h β-conidia i, k, l α-phialides j α-conidia. Scale Bars: 2 cm (a, c, d); 0.5 cm (b); 20 μm (e–h, j); 10 μm (i, k. l).

https://binary.pensoft.net/fig/1050582 Host.

Parasitic on Ophiocordyceps sp. (Ophiocordycipitaceae).

 Distribution.

China, Yunnan Province.

 Material examined.

China, Yunnan Province, Xishuangbanna Dai Autonomous Prefecture, Jinghong City, parasitic on Ophiocordyceps sp. (Ophiocordycipitaceae), on insects buried in soil, with erect synnemata, 23°47′9″N, 102°51′41″E, alt. 2053 m, 25 September 2022, D.X. Tang. Paratypes: YHH 2206010; other collections: YHH 2207049; YHH 2206053. Culture ex-type: YFCC 02959283; Other cultures: YFCC 02959284, YFCC 02959285, YFCC 02959286.

 Notes.

Polycephalomyces jinghongensis was sister to Po. multiperitheciatae (Fig. 1: BS = 100%, BPP = 1.00). However, Po. multiperitheciatae differs by 6/556 bp in ITS, 3/898 bp in SSU, 2/829 bp in LSU, 23/913 bp in TEF-1α, 4/679 bp in RPB2 from Po. jinghongensis. Po. jinghongensis was distinct from other species of Polycephalomyces by the white to orange-yellow colour of the caespitose synnemata (Table 3). Thus, Po. jinghongensis was introduced as a new species under the genus Polycephalomyces.

Morphology of asexual morph species of the genus Polycephalomyces.

Species Host Synnemata Phialides Conidia References
Po. albiramus Gryllotalpa sp. (Orthoptera, Gryllotalpidae) Stipitate, gathered, branched, white to pale yellow, numerous, cylindrical and tapering at the apex, without fertile head Phialides narrowly subulate, awl-shaped Conidia cylindrical to obovoid or subglobose Xiao et al. (2023)
Po. baltica Nymph or short-winged female bark louse (Psocoptera: Troctopsocidae) Synnemata, simple, roundish Phialides, light colored, micronematous, flask-shaped Conidia globose, catenulate Poinar and Vega (2020)
Po. cylindrosporus Coleoptera, Formicidae and Pentatomidae Synnemata cylindrical to capitate, stipitate, slender, branched Phialides on verticils and/or acropleurogenously forming loosely arranged flared hymenia Conidia one-type, cylindrical to bacilliform Matočec et al. (2014)
Po. ditmarii Paravespula vulgaris (Wasp) Synnemata 2 to 3 distinct branches, yellowish to white, darkening at the base; surmounted by a small subsurface capitulum, dotted with numerous small blisters of orange-yellow colour Phialides elongate, cylindrical, attenuating at the apex globose to subglobose Van Vooren and Audibert (2005)
Po. formosus (Type) Coleoptera larvae or Ophiocordyceps barnesii Synnemata 2 long, gathered, branched, with cylindrical stipe, with fertile head, spherical, white cylindrical, tapering gradually Conidia one-type, ellipsoid or ovoid Kobayasi (1941)
In culture (PDA) Synnemata 2–3 branches,arising as several radiating rings on the colony Phialides terminal parts of Synnemata, cylindrical to subulate at the base; Conidia of one type, one-celled, smooth-walled, ellipsoid to ovoid, arising in a conidial mass on the agar or on the terminal portions of synnemata Wang et al. (2021)
In slide culture Phialides monothetic and solitary or acropleurogenous in the whorls of 1–4, narrowly lageniform or subulate Conidia obovoid to oblong ellipsoidal or cylindrical, forming irregular spore balls near the apex of phialides Wang et al. (2021)
Po. ramosus Lepidoptera larvae or Hirsutella guignardii Synnemata solitary, crowded or caespitose, unbranched or branched, conidial mass yellow to orange-yellow α-phialides cylindrical to narrowly lageniform; β-phialides narrowly lageniform or subulate α-conidia, ovoid; β-conidia, fusiform Seifert (1985); Bischof et al. (2003)
Po. paludosus Lepidoptera larva Capitate, cinnamon brown, branched, the branches at right angles Subulate, phialides occurring scattered on the branches below the heads, ventricose, occasionally stellate above Conidia produced singly, hyaline, obovoid, covered by agglutinated mucus Mains (1948)
Po. tomentosus Myxomycetes Fructification a synnemata Conidia three-type, globose or ellipsoidal or cylindrical Seifert (1985)
Po. jinghongensis Ophiocordyceps sp. (Ophiocordycipitaceae) Synnemata caespitose, unbranched or branched, white to orange-yellow colour α-phialides verticillate and acropleurogenous on conidiophores,and solitary on hyphae; lanceolate. β-phialides acropleurogenous in whorls of 2–3 or intercalary and terminal on conidiophores and solitary on hyphae; diamond-shaped. α-conidia oval or long oval shape, β-conidia cylindrical This study
Po. multiperitheciatae Ophiocordyceps multiperitheciata Synnemata white to pale yellow, numerous, branched, with fertile head α-phialides verticillate and acropleurogenous on conidiophores, and solitary on hyphae; spear point. β-phialides acropleurogenous in whorls of 2–3 or intercalary and terminal on conidiophores and solitary on hyphae; subulate. α-conidia oval β-conidia linear This study
Po. myrmecophilus Ophiocordyceps acroasca and Ophiocordyceps sp. Absent α-phialides verticillate and acropleurogenous on conidiophores, and solitary on hyphae; lanceolate, β-phialides acropleurogenous in whorls of 2–3 or intercalary and terminal on conidiophores and solitary on hyphae; sickle shape. α-conidia round or ovoid; β-conidia, elliptical This study
Fungi Hypocreales Ophiocordycipitaceae 799A5657-2B7C-5256-858B-AA094FBD291D Polycephalomyces multiperitheciatae 851499 Hong Yu bis, Z.H. Liu & D.X. Tang sp. nov. Fig. 4 Etymology.

The species name referred to the host species, Ophiocordyceps multiperitheciata.

 Diagnosis.

Polycephalomyces multiperitheciatae are similar to that of Po. jinghongensis regarding the production of α-conidia oval, but Po. jinghongensis differ by being parasitic on O. multiperitheciata, synnemata clustered, white, β-conidia, linear.

 Holotype.

China, Yunnan Province, Honghe Hani and Yi Autonomous Prefecture, Yuanyang County, parasitic on Ophiocordyceps multiperitheciata (Ophiocordycipitaceae), on insects buried in soil, with erect stromata, 22°1′51″N, 100°52′42″E, alt. 703 m, 25 September 2022, Hong Yu bis (YHH 2206031).

 Sexual morph.

Undetermined.

 Asexual morph.

Synnemata arising from the stromata of Ophiocordyceps multiperitheciata, 0.8–1.8 cm long 0.2–0.5 cm thick, clustered, white to pale yellow, numerous, branched, with fertile head. Colonies on PDA growing slowly, attaining a diameter of 1.8–2.1 cm in 3 weeks at 25 °C, clustered, white and reverse dry yellow. Synnemata emerging after 15 days, solitary, branched and 0.8–2.1 cm long, showing radiating distributions. Phialides existing in two types: α- and β-phialides. Both types of phialides often reproduce new conidia at their own apices or sides, collarettes not flared, periclinal thickening not visible. α-phialides verticillate and acropleurogenous on conidiophores and solitary on hyphae; spear point, tapering gradually from the base to the apex, 10.5–18.7 μm long, 1.1–1.9 μm wide at the base and 0.4–0.6 μm wide at the apex. β-phialides acropleurogenous in whorls of 2–3 or intercalary and terminal on conidiophores and solitary on hyphae; subulate, tapering abruptly from the base to the apex, 11.3–28.8 μm long, 1.2–2.5 μm wide at the base and 0.5–1.1 μm wide at the apex. α-conidia,oval and occurring in the conidial mass on the agar or on the final portion of synnemata, 0.6–1.1 × 0.3–0.6 μm; β-conidia, linear and produced on the surface mycelium of colony, multiple, usually formed as spore balls at the phialidic apex, 0.8–1.3 × 0.3–0.7 μm.

10.3897/mycokeys.105.119893.figure4 4CB9B1FA-1CDA-5A61-8255-196D02F4FA70

Morphological features of Polycephalomyces multiperitheciatae (Holotype: YHH 2206047) a overview of Polycephalomyces multiperitheciatae and its host b Synnemata on the insect c, d colony obverse and reverse g, j α-phialides e, f, h, i β-phialides k α-conidia l β-conidia. Scale Bars: 2 cm (a, c, d); 0.6 cm (b); 20 μm (e–i, k); 50 μm (j); 10 μm (l).

https://binary.pensoft.net/fig/1050583 Host.

Parasitic on Ophiocordyceps multiperitheciata (Ophiocordycipitaceae).

 Distribution.

China, Yunnan Province.

 Material examined.

China, Yunnan Province, Honghe Hani and Yi Autonomous Prefecture, Yuanyang County, parasitic on Ophiocordyceps multiperitheciata (Ophiocordycipitaceae), on insects buried in soil, with erect stromata, 22°1′51″N, 100°52′42″E, alt. 703 m, 25 September 2022, D.X. Tang. Paratypes: YHH 2209032; other collections: YHH 2209033; YHH 2209034. Culture ex-type: YFCC 06149287; Other cultures: YFCC 06149288, YFCC 06149289, YFCC 06149290, YFCC 06149291, YFCC 06149292.

 Notes.

Polycephalomyces multiperitheciatae is sister to Po. jinghongensis (Fig. 1: BS = 100%, BPP = 1.00). Po. multiperitheciatae is distinct from other species of Polycephalomyces, parasitising Ophiocordyceps multiperitheciata synnemata clustered, with fertile head, β-conidia, linear (Table 3). Thus, Po. multiperitheciatae was introduced as a new species under the genus Polycephalomyces.

 Fungi Hypocreales Ophiocordycipitaceae E26D633A-6463-540C-AA06-BFF183F4C7BA Polycephalomyces myrmecophilus 851500 Hong Yu bis, Z.H. Liu & D.X. Tang sp. nov. Fig. 5 Etymology.

myrmecophilus = myrmecophilous, the epithet referred to the species parasitising myrmecophilous Ophiocordyceps species.

 Diagnosis.

Polycephalomyces myrmecophilus are similar to that of Po. ramosus regarding the production of two types of conidia, but Po. myrmecophilus differ by α-conidia round or ovoid, β-conidia elliptical.

 Holotype.

China, Yunnan Province, Pu’er City, The Sun River National Forest Park, parasitic on Ophiocordyceps acroasca (Ophiocordycipitaceae), on insects underside of leaves, with erect stromata, 30°34′34″N, 101°6′24″E, alt. 1095 m, 28 September 2020, Hong Yu bis (YHH 2009001);

 Sexual morph.

Undetermined.

 Asexual morph.

Synnemata arising from the Ophiocordyceps acroasca or Colobopsis sp. corpses, tomentose, white. Colonies on PDA growing slowly, attaining a diameter of 1.7–2.1 cm in 3 weeks at 25 °C, villous, cinerous, and reverse black yellow. Phialides existing in two types: α- and β-phialides. Both types of phialides often reproduce new phialides at their own apices, collarettes not flared, periclinal thickening not visible. α-phialides verticillate and acropleurogenous on conidiophores and solitary on hyphae; lanceolate, tapering gradually from the base to the apex, 6.1–14.5 μm long, 1.4–2.3 μm wide at the base and 0.8–1.8 μm wide at the apex. β-phialides acropleurogenous in whorls of 2–3 or intercalary and terminal on conidiophores and solitary on hyphae; sickle-shaped, tapering abruptly from the base to the apex, 9.8–17.6 μm long, 0.9–1.6 μm wide at the base and 0.4–1.1 μm wide at the apex. α-conidia round or ovoid, and occurring in the conidial mass on the agar or on the final portion of synnemata, 0.4–0.9 × 0.3–0.9 μm; β-conidia elliptical and produced on the surface mycelium of colony, single or multiple, usually in the form of spore balls at the phialidic apex, 0.6–1.3 × 0.3–0.8 μm.

10.3897/mycokeys.105.119893.figure5 C00BB315-3FD6-5B47-B9A9-B37D4A32FF5C

Morphological features of Polycephalomyces myrmecophilus (Holotype: YHH 2009001) a overview of Polycephalomyces myrmecophilus and its host b, c colony obverse and reverse d α-phialides f–h β-phialides e α-conidia i β-conidia. Scale Bars: 2 cm (a–c); 20 μm (d–h); 10 μm (i).

https://binary.pensoft.net/fig/1050584 Host.

Parasitic on Ophiocordyceps acroasca and Ophiocordyceps sp.

 Distribution.

China, Yunnan Province.

 Material examined.

China, Yunnan Province, Pu’er City, The Sun River National Forest Park, parasitic on Ophiocordyceps acroasca (Ophiocordycipitaceae), on insects underside of leaves, with erect stromata, 30°34′34″N, 101°6′24″E, alt. 1095 m, 28 September 2020, D.X. Tang. Paratype: YHH 2006020. Culture ex-type: YFCC 09289443; Other cultures: YFCC 09289444.

 Notes.

Polycephalomyces myrmecophilus was sister to Cordyceps pleuricapitata (Fig. 1: BS = 100%, BPP = 1.00). Po. myrmecophilus was distinct from other species of Polycephalomyces, being parasitic on Ophiocordyceps acroasca and Ophiocordyceps sp. and producing β-phialides sickle-shaped, α-conidia round or ovoid, β-conidia elliptical (Table 3). Thus, Po. myrmecophilus was introduced as a new species under the genus of Polycephalomyces.

 Discussion

Our taxonomic investigations revealed four new species of the family Polycephalomycetaceae, Pl. litangensis, Po. jinghongensis, Po. multiperitheciatae and Po. myrmecophilus. Morphological observations suggested that four species have sufficient morphological differences to justify their segregation into four species. A new species, Pl. litangensis, was described in the genus Pleurocordyceps. Pleurocordyceps litangensis was similar to Pl. agaricus, Pl. aurantiacus, Pl. lanceolatus, Pl. marginaliradians, Pl. sinensis, Pl. vitellina, Pl. yunnanensis, Pl. nutansis and Pl. heilongtanensis, by producing two types of conidia, while Pl. Parvicapitata and Pl. lianzhouensis had only one type of conidia. Pl. litangensis was distinct from other species of Pleurocordyceps, with having α-phialides spear point, β-phialides subulate, α-conidia ovoid or elliptic. Moreover, Pl. litangensis and Pl. sinensis both had the same host (O. sinensis) and β-Conidia, but their phialides, α-conidia size and shape were different (Table 2). Herein, we described three new species, namely, Po. jinghongensis, Po. multiperitheciatae and Po. myrmecophilus, enriching the species diversity in the genus Polycephalomyces. 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), Po. paludosus (Mains 1948), Po. ramosus (Seifert 1985; Bischof et al. 2003) and Po. tomentosus (Seifert 1985). These species either lacked molecular data or their updated strain descriptions did not match those of the protologue (Wang et al. 2021). These three new species were similar to Po. ramosus, producing two types of conidia, while Po. baltica, Po. cylindrosporus, Po. ditmarii, Po. paludosus and Po. albiramus (Xiao et al. 2023) had only one type of conidia. Po. jinghongensis was distinct from Po. ramosus, being parasitic on Ophiocordyceps sp. producing longer α-conidia oval or long oval shape and β-conidia columns. Po. multiperitheciatae differed from Po. ramosus, being parasitic on O. multiperitheciata, having synnemata with fertile head and β-conidia linear. Po. myrmecophilus was distinguished from Po. ramosus, being parasitic on the fungus O. acroasca, producing synnemata, α-conidia round or ovoid, and β-conidia elliptical, without producing synnemata from the colonies, whereas Po. ramosus was parasitic on Lepidoptera larvae or Hirsutella guignardii, with α-conidia ovoid and β-conidia fusiform (Table 3).

Some species of the family Polycephalomycetaceae have been reported from more than one host, indicating their non-host specific nature (Bischof et al. 2003; Wang et al. 2012, 2015a, b; Matočec et al. 2014; Crous et al. 2017; Xiao et al. 2018). Pl. lianzhouensis (Wang et al. 2014) was found to parasitise insects along with the species of the genus Ophiocordyceps. The field investigation and studies showed that Pl. litangensis also parasitised O. sinensis, a phenomenon known as hyperparasitism. Most species of the genus Polycephalomyces parasitise insects in the orders Coleoptera and Hemiptera, and we have already discovered that Po. jinghongensis, Po. multiperitheciatae and Po. myrmecophilus are hyperparasitic on the species of Ophiocordyceps, expanding the diversity of hosts in Polycephalomyces. In subsequent studies, we should delve deeper into the ecological habits and hyperparasitic phenomena of the family Polycephalomycetaceae, explore the evolutionary relationship between hyperparasitic species and entomophytic fungi and promote their development and utilisation.

Xiao et al. (2023) introduced Pl. nutansis as a new species under the genus Pleurocordyceps. However, Pl. sinensis and Pl. nutansis were found to be grouped together in the phylogenetic tree, which may be the reason and they are sister taxa to each other. Similarly, molecular phylogenetic analysis has shown that Pl. nipponica and Pl. kanzashianus are clustered together. Nevertheless, Wang et al. (2021) pointed out that they were distinct species, based on their sexual morphology characteristics. In addition, Wang et al. (2021) noted the description of the spore type of Pl. lianzhouensis was not clear and future research should strengthen the observation of its asexual morphology to determine its more accurate classification position. Cordyceps pleuricapitata has formed a monophyletic branch in the genus Polycephalomyces. Xiao et al. (2023) noted 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, it was not possible to clarify the precise position of C. pleuricapitata and its classification at this time. These classifications issues require further research. Phylogeny based on our concatenated data also supported that our four new species belonged to the family Polycephalomycetaceae and were distinct from each other (Fig. 1). Four strains, namely, Pleurocordyceps sp. NBRC109990, Pleurocordyceps sp. NBRC109987, Pleurocordyceps sp. NBRC110224 and Pleurocordyceps sp. NBRC109988 and Pl. litangensis were aggregated into one branch. However, the four strains had only LSU sequences in the NCBI database and were classified as undefined species in Pleurocordyceps incertae sedis. Future research will require additional morphological and phylogenetic work to clarify their taxonomic status.

 Acknowledgements

We thank the National Natural Science Foundation of China (No. 31760011). We thank all those who have provided assistance for this work. Participation and sponsorship of the Yunnan University Professional Degree Graduate Practice Innovation Fund Program (ZC-22222937).

 Additional information Conflict of interest

The authors have declared that no competing interests exist.

 Ethical statement

No ethical statement was reported.

 Funding

This work was supported by National Natural Science Foundation of China (No.31760011). Participation and sponsorship of the Yunnan University Professional Degree Graduate Practice Innovation Fund Program (ZC-22222937).

 Author contributions

Zuoheng Liu: Mainly responsible for article conception, writing and editing and also mainly responsible for species identification (contributed equally to this work); Dexiang Tang: Mainly responsible for article conception writing and editing, morphological analysis and phylogenetic analysis(contributed equally to this work); Yingling Lu: Mainly responsible for article conception, and also responsible for experimental guidance and design; Responsible for the language polishing and format modification.Juye Zhu: Collecting the information of specimens and GenBank entry number required for research. Lijun Luo; Tao Sun: Responsible for picture editing and processing. Hong Yu: Investigation, responsible for the review and modification of the article, and conducting project administration and supervision.

 Author ORCIDs

Zuoheng Liu https://orcid.org/0000-0003-4118-3694

Dexiang Tang https://orcid.org/0000-0002-7662-224X

Yingling Lu https://orcid.org/0009-0008-8119-1975

Juye Zhu https://orcid.org/0000-0002-4184-5646

Lijun Luo https://orcid.org/0000-0002-1709-0781

Tao Sun https://orcid.org/0000-0001-7837-2101

Hong Yu https://orcid.org/0000-0002-2149-5714

 Data availability

All of the data that support the findings of this study are available in the main text.

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