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Research Article
Interesting mycoparasites and Paradingleyomyces lepidopterorum gen. et sp. nov. (Hypocreales, Polycephalomycetaceae) from Yunnan Province, China
expand article infoYi Wang, De-Ping Wei, Xing-Can Peng§, Ji-Chuan Kang, Zeng-Zhi Li|, Chun-Ru Li|, Xian Zhang§, Gui-Ying Wang, Yun Zhou, Xin-Sheng He, Putarak Chomnunti§, Ting-Chi Wen
‡ Guizhou University, Guiyang, China
§ Mae Fah Luang University, Chiang Rai, Thailand
| Zhejiang BioAsia Life Science Institute, Pinghu, China
¶ Southwest University of Science and Technology, Mianyang, China
Open Access

Abstract

A novel genus, Paradingleyomyces was introduced to accommodate Pa. lepidopterorum sp. nov., based on a multigene phylogenetic analysis and its distinct morphological characteristics. Maximum likelihood (ML) and Bayesian inference analyses (BI) of ITS, SSU, LSU, tef-1α, rpb1, and rpb2 sequence data shown that Pa. lepidopterorum formed an independent lineage nested between Perennicordyceps and Dingleyomyces. Morphologically, Paradingleyomyces is distinguished from Perennicordyceps by the presence of a white subiculum on the stromata of Ophiocordyceps cf. cochlidiicola. Perithecia are produced sporadically from the base to the apex of the stromata, and the secondary ascospores exhibit a notable length-to-width ratio. These characteristics distinguish Paradingleyomyces from Perennicordyceps which exhibits tortuous, branched, clavate to cylindrical stromata with rhizomorphs, parasitism of coleopteran and hemipteran larvae, and colonizes a broader range of fungal hosts. Additionally, perithecia in Perennicordyceps typically arise from the middle to the upper regions of the stromata, with secondary ascospores displaying a comparatively lower length-to-width ratio. Paradingleyomyces is morphologically identical to Dingleyomyces in its direct production of superficial perithecia on the stromata of Ophiocordyceps species. However, the phylogenetic analysis indicates that Paradingleyomyces and Dingleyomyces are not congeneric. Moreover, this study introduces another novel species, Polycephalomyces tengchongensis, and a novel sexual morph of Pleurocordyceps yunnanensis. Dimorphic phialides and conidia of Pleurocordyceps parvicapitata were observed and described for the first time based on a fresh collection from Tengchong County, Yunnan Province, China.

Key words

Entomopathogenic fungi, new genus, phylogeny, taxonomy

Introduction

Polycephalomyces was introduced as an entomopathogenic genus by Kobayasi (1941), based on the asexual morph of the Po. formosus, which was characterized by polycephalous synnemata with white to pale yellow conidial masses on the tips. Species of Polycephalomyces form parasitic associations with a wide range of hosts, including insects, fungi and myxomycetes (Shrestha et al. 2017; Xiao et al. 2023). Among these, Ophiocordyceps species are the most common hosts to Polycephalomyces. For instance, Polycephalomyces sinensis was found as a hyperparasite on Ophiocordyceps sinensis, Po. ramosus on Hirsutella guignardii and Pleurocordyceps lianzhouensis on O. crinalis (Massee 1895; Kobayasi 1941; Kepler et al. 2013; Wang et al. 2014). Members of Polycephalomyces were expanded to include sexual species that were phylogenetically distant from the Ophiocordyceps sensu stricto by Kepler et al. (2013). In this study, the sexual morph of Polycephalomyces was described as possessing firm, pliant, multifurcating stromata, perithecia that are either superficial or immersed in an apical or subapical pulvinate cushion, filiform asci and disarticulating ascospores. The taxonomic placement of Polycephalomyces remained uncertain until Quandt et al. (2014) classified it within the family Ophiocordycipitaceae based on phylogenetic analyses of combining SSU, LSU, tef-1α, rpb1 and rpb2 sequence. Perennicordyceps was later established by Matočec et al. (2014) to accommodate four species (Perennicordyceps cuboidea, Pe. paracuboidea, Pe. prolifica, and Pe. ryogamiensis), which were previously classified under Polycephalomyces, based on comprehensive morphological characteristics and molecular data analyses. Perennicordyceps is characterized by the presence of superficial perithecia and acremonium-like or hirsutella-like asexual morphs (Matočec et al. 2014; Hyde et al. 2018; Wei et al. 2022). Wang et al. (2021) established Pleurocordyceps to accommodate ten species that previously were placed in Polycephalomyces. An increasing number of polycephalomyces-like species have been added to Polycephalomyces, Perennicordyceps, and Pleurocordyceps, contributing to a more refined understanding of their natural classification and phylogenetic relationships (Wang et al. 2014; Wang et al. 2015a, 2015b; Liang et al. 2016; Crous et al. 2017a; Xiao et al. 2018, 2023; Yang et al. 2020). Xiao et al. (2023) constructed a backbone tree of Hypocreales using extensive taxon sampling, and the result clearly showed that Polycephalomyces, Perennicordyceps, and Pleurocordyceps form a monophyletic clade distinct from Ophiocordycipitaceae. As a result, a new family, Polycephalomycetaceae, was established to accommodate these three genera. It is worth noting that in the phylogenetic analysis conducted by Xiao et al. (2023), Polycephalomycetaceae was identified as a sister clade with Ophiocordycipitaceae. However, in the study conducted by Wei et al. (2022), Polycephalomycetaceae was found to form a sister clade to Clavicipitaceae. These contrasting findings suggest that the phylogenetic relationship between Polycephalomycetaceae and other hypocrealean families requires further confirmation through future research, incorporating more comprehensive taxon sampling. Therefore, discovering hidden or yet unknown species within Polycephalomycetaceae is essential for improving our understanding of this family’s evolutionary position.

During our ongoing exploration of the diversity of entomopathogenic fungi and their associated fungi in Yunnan Province, China, several polycephalomyces-like species were found from various hosts including Elaphomyces sp., lepidopteran larvae, Ophiocordyceps nutans, and Perennicordyceps cf. elaphomyceticola. This study aims to assess the phylogenetic relationships of these samples with existing species of Polycephalomycetaceae using a concatenated SSU, ITS, LSU, tef-1α, rpb1 and rpb2 sequences, as well as detailed morphological analyses. The morphological observations and phylogenetic analyses allowed us to introduce a new genus, Paradingleyomyces, a new species, Polycephalomyces tengchongensis, a new sexual morph of Pleurocordyceps yunnanensis, and a new collection of Pleurocordyceps parvicapitata. These findings expand our understanding of this unique group of entomopathogens and mycoparasites, offering fresh and novel insights into their morphology, ecology and evolutionary relationships.

Materials and methods

Morphological study and isolation

To explore the diversity of fungal resources, samples were collected from tropical and subtropical forests rich in evergreen trees diversity in southwestern China. Morphological studies followed the guidelines proposed by Senanayake et al. (2020). Specimens were placed in small plastic boxes and transported to the laboratory for isolation. Both specimens and colonies were photographed using a Canon 6D camera equipped with a 100 mm MACRO lens for detailed morphological documentation. Fruiting bodies were examined, and free-hand sections were prepared using a stereomicroscope (Leica S9E). Slides containing sectioning of the fertile parts were mounted for microscopic observation with a Leica DM2500 compound microscope equipped with a digital camera. Micro-morphological characters, including ascomata, perithecia, peridium texture, asci, ascospores, secondary ascospores, conidiophores, phialides, and conidia were photographed and measured by using the Leica microsystem for precise documentation and analysis. A small mass of tissue from the fertile parts of the fungus or insect bodies was transferred to potato glucose agar (PDA) plate using sterile inoculation needles and incubated at 25 °C to obtain pure isolates (Wen et al. 2014; Aini et al. 2020; Peng et al. 2024). Freshly collected specimens were dried using silica gel to preserve them as dry specimens. The cultures were deposited in the Herbarium of Kunming Institute of Botany Culture Collection (KUNCC; http://english.kib.cas.cn/) and the dry specimens were deposited in the Herbarium of Cryptogams, Kunming Institute of Botany of the Chinese Academy of Sciences (KUN; http://kun.kingdonia.org/).

DNA extraction, PCR amplification and sequencing

DNA was extracted from fresh specimens and cultures using the E.Z.N.A.TM Fungal DNA MiniKit (Omega Biotech, CA, USA) following the manufacturer’s protocols. Polymerase chain reaction (PCR) was performed to amplify six loci: the small subunits nuclear of rDNA (SSU), the internal transcribed spacer (ITS), the large subunit nuclear of rDNA (LSU), the transcription elongation factor-1α (tef-1α), the partial RNA polymerase II largest subunit (rpb1) and the partial RNA polymerase II second largest subunit (rpb2). The primer pairs used for amplifying the six loci were as follows: NS1 and NS4 for SSU (White et al. 1990), ITS5 and ITS4 for ITS (White et al. 1990), LROR and LR5 for LSU (Vilgalys and Hester 1990), 983F and 2218R for tef-1α (Rehner and Buckley 2005), CRPB1A and RPB1Cr for rpb1 (Castlebury et al. 2004), and RPB2-5F and RPB2-7R for rpb2 (Liu et al. 1999). Amplification reaction was performed in a 50 μL reaction volume containing 4 μL of DNA template, 2 μL of each forward and reverse primers (10 pM), 22 μL of 2× Taq PCR StarMix with Loading Dye (GenStar) and 20 μL of twice-sterilized water. The amplification conditions for ITS, LSU, SSU, tef-1α, rpb1, and rpb2 were as follows: (1) 3 min at 94 °C, (2) 33 cycles of denaturation at 94 °C for 30 s, annealing (ITS at 52 °C for 50 s, SSU at 47 °C for 1 min 20 s, LSU at 50 °C for 30 s, tef-1α at 58 °C for 50 s, rpb1 and rpb2 at 51 °C for 40 s), and elongation (ITS at 72 °C for 45 s, SSU and LSU at 72 °C for 1min 50 s, tef-1α at 72 °C for 1 min, rpb1 and rpb2 at 72 °C for 1 min 20 s), and (3) final extension at 72 °C for 10 min. The PCR products were sent to Tsingke Biological Technology in Chongqing, China, for sequencing, and the resulting sequences were submitted to GenBank for the assignment of accession numbers.

Phylogenetic analysis

The newly generated sequences were checked and assembled using BioEdit v.7.0.5.3 (Hall et al. 2011). The assembled sequences were then subjected to BLAST searches in the GenBank database of National Center for Biotechnology Information (NCBI) to confirm their identities. Taxa used for phylogenetic analyses were chosen based on relevant publications and presented in Table 1. The individual gene was aligned using MAFFT (Katoh and Standley 2013). Trimal v1.2 was used to remove alignments sites that did not achieve a user specified gap score of 0.6 (Capella-Gutiérrez et al. 2009). The trimmed alignments were concatenated using FasParser 2.10.0 (Sun 2017). The final combined alignment was subjected to Maximum likelihood (ML) and Bayesian inference (BI) analyses. ML analysis was performed using IQ-TREE 1.6.12, with branch support estimated from 1000 ultrafast bootstraps replicates (Minh et al. 2020).MrModelTest v. 2.3 (Nylander 2004) was used to determine the best evolutionary model for Bayesian inference analysis according to the Akaike Information Standard (AIC). The best-fit models GTR+I+G, were determined for SSU, ITS, LSU, tef1-α, rpb1, and rpb2. The BI analysis was carried out using MrBayes on XSEDE (3.2.7a) through the CIPRES Science Gateway V 3.3 platform (Miller et al. 2010). Four Markov chain Monte Carlo (MCMC) simulations were run for 50,000,000 generations, sampling every 1000 generations and discarding the first 25% as burn-in. The remaining trees were used to calculate Bayesian posterior probabilities. The resulting trees were visualized using FigTree v1.4.3 (Rambaut 2012). To determine whether the taxa represented new species or new records, the guidelines of Jeewon and Hyde (2016) were followed.

Table 1.

Accession numbers of taxa used in this study. Newly generated sequences are indicated in bold. T Represents type strain, type specimens or neotype.

Current name Voucher SSU ITS LSU tef-1α rpb1 rpb2 Reference
Dingleyomyces lloydii PDD1212154T OR647563 OR602634 OR602640 OR588853 OR588860 OR588858 Johnston and Park (2023)
Paradingleyomyces lepidopterorum HKAS 131926 T OR878363 OR828238 OR829674 OR880683 This study
Paradingleyomyces lepidopterorum HKAS 131927 OR878364 OR828239 OR880679 OR829675 This study
Paradingleyomyces lepidopterorum HKAS 131921 OR828242 OR829678 This study
Perennicordyceps cuboidea NBRC 103836 JN941721 JN943332 JN941420 AB972951 JN992455 AB972955 Schoch et al. (2012)
Perennicordyceps cuboidea NBRC 101740 JN941724 JN943331 JN941417 KF049684 JN992458 Schoch et al. (2012)
Perennicordyceps cuboidea TNS-F-18487 KF049609 KF049628 KF049683 Kepler et al. (2013)
Perennicordyceps cuboidea NBRC 101739 AB378668 AB378649 Ban et al. (2009)
Perennicordyceps elaphomyceticola NTUCC 17-022 MK840824 MK840813 MK839230 MK839221 MK839212 Yang et al. (2020)
Perennicordyceps elaphomyceticola MFLU 21-0262 OQ172101 OQ172064 OQ172032 OQ459718 OQ459747 OQ459792 Xiao et al. (2023)
Perennicordyceps elaphomyceticola MFLU 21-0263 OQ172102 OQ172065 OQ172033 OQ459719 OQ459748 OQ459793 Xiao et al. (2023)
Perennicordyceps elaphomyceticola MFLU 21-0264 OQ172103 OQ172067 OQ172035 OQ459720 OQ459749 OQ459794 Xiao et al. (2023)
Perennicordyceps elaphomyceticola MFLU 21-0266 OQ172112 OQ172068 OQ172036 OQ459732 OQ459760 OQ459806 Xiao et al. (2023)
Perennicordyceps elaphomyceticola KUNCC23-16074 PP129613 OR878367 OR828243 OR829679 This study
Perennicordyceps lutea KUMCC 3004 OQ474910 Xiao et al. (2023)
Perennicordyceps paracuboidea NBRC 100942 JN941711 JN943337 JN941430 AB972954 JN992445 AB972958 Schoch et al. (2012)
Perennicordyceps paracuboidea NBRC 101742 JN941710 JN943338 JN941431 KF049685 JN992444 KF049669 Schoch et al. (2012)
Perennicordyceps prolifica NBRC 100744 JN941709 AB925942 JN941432 AB972952 JN992443 AB972956 Ban et al. (2015)
Perennicordyceps prolifica NBRC 101750 JN941708 JN943340 JN941433 AB972953 JN992442 AB972957 Ban et al. (2015)
Perennicordyceps prolifica TNS-F-18547 KF049613 KF049660 KF049632 KF049687 KF049649 KF049670 Kepler et al. (2013)
Perennicordyceps prolifica NBRC 103839 JN941706 JN943342 JN941435 JN992440 Schoch et al. (2012)
Perennicordyceps prolifica NBRC 103838 JN941707 JN943339 JN941434 JN992441 Schoch et al. (2012)
Perennicordyceps prolifica TNS-F-18481 KF049612 KF049659 KF049631 KF049686 KF049648 Kepler et al. (2013)
Perennicordyceps prolifica AB027324 AB027370 Nikoh and Fukatsu. (2000)
Perennicordyceps ryogamiensis NBRC 103842 JN941701 JN943345 JN941440 JN992435 Schoch et al. (2012)
Perennicordyceps ryogamiensis NBRC 101751 JN941703 JN943343 JN941438 KF049688 JN992437 Schoch et al. (2012)
Pleurocordyceps agarica YHHPA1305T KP276655 KP276651 KP276659 KP276663 KP276667 Wang et al. (2015a, b)
Pleurocordyceps agarica YHCPA1303 KP276657 KP276653 KP276661 KP276665 KP276669 Wang et al. (2015a, b)
Pleurocordyceps agarica YHCPA1307 KP276658 KP276654 KP276662 KP276666 KP276670 Wang et al. (2015a, b)
Pleurocordyceps aurantiacus MFLUCC 17-2113T MG136904 MG136916 MG136910 MG136874 MG136866 MG136870 Xiao et al. (2018)
Pleurocordyceps aurantiacus MFLU 17-1393T MG136907 MG136919 MG136913 MG136877 MG136868 MG136873 Xiao et al. (2018)
Pleurocordyceps aurantiacus MFLU 21-0276 OQ172097 OQ172072 OQ172042 OQ459714 OQ459788 Xiao et al. (2023)
Pleurocordyceps aurantiacus GACP 20-2306 OQ172098 OQ172069 OQ172041 OQ459715 OQ459789 Xiao et al. (2023)
Pleurocordyceps formosus ARSEF 1424 KF049615 KF049661 KF049634 KF049689 KF049651 KF049671 Kepler et al. (2013)
Pleurocordyceps heilongtanensis KUMCC 3008 OQ172111 OQ172091 OQ172063 OQ459731 OQ459759 OQ459805 Xiao et al. (2023)
Pleurocordyceps kanzashianus AB027325 AB027371 AB027371 Kepler et al. (2013)
Pleurocordyceps lanceolatus GACP 17-2004T OQ172110 OQ172076 OQ172046 OQ459726 OQ459754 OQ459800 Xiao et al. (2023)
Pleurocordyceps lanceolatus GACP 17-2005T OQ172109 OQ172077 OQ172047 OQ459727 OQ459755 OQ459801 Xiao et al. (2023)
Pleurocordyceps lianzhouensis HIMGD20918 KF226245 EU149921 KF226246 KF226248 KF226247 Zhang et al. (2007)
Pleurocordyceps lianzhouensis GIMYY9603 KF226249 EU149922 KF226250 KF226252 KF226251 Zhang et al. (2007)
Pleurocordyceps marginaliradians MFLUCC 17-2276 MG136909 MG136921 MG136915 MG136879 MG271930 Xiao et al. (2018)
Pleurocordyceps marginaliradians MFLU 17-1582 MG136908 MG136920 MG136914 MG136878 MG136869 MG271931 Xiao et al. (2018)
Pleurocordyceps nipponicus BCC 1881 KF049618 KF049636 KF049692 KF049674 Kepler et al. (2013)
Pleurocordyceps nipponicus NHJ 4268 KF049621 KF049639 KF049695 KF049654 KF049676 Kepler et al. (2013)
Pleurocordyceps nipponicus BCC 2325 KF049622 KF049665 KF049640 KF049696 KF049655 KF049677 Kepler et al. (2013)
Pleurocordyceps nipponicus BCC 18108 MF416624 KF049657 MF416569 MF416517 MF416676 MF416462 Kepler et al. (2013)
Pleurocordyceps nipponicus NBRC 101408 JN941751 JN943303 JN941390 JN992485 Schoch et al. (2012)
Pleurocordyceps nipponicus NBRC 101407 JN941752 JN943302 JN941389 JN992486 Schoch et al. (2012)
Pleurocordyceps nipponicus NBRC 101406 JN941753 JN943301 JN941388 JN992487 Schoch et al. (2012)
Pleurocordyceps nipponicus Cod-RE1202 MG031286 KX827757 MG031248 MG196133 MG196175 Sangdee et al. (2017)
Pleurocordyceps nipponicus BCC 1682 KF049620 KF049664 KF049638 KF049694 Kepler et al. (2013)
Pleurocordyceps nutansis MFLU 21-0275T OQ172119 OQ172073 OQ172048 Xiao et al. (2023)
Pleurocordyceps nutansis GACP 19-1906 OQ172117 OQ172079 OQ172049 Xiao et al. (2023)
Pleurocordyceps onorei BRA: CR23902T KU898841 Crous et al. (2017a)
Pleurocordyceps onorei BRA: CR23904 KU898843 Crous et al. (2017a)
Pleurocordyceps parvicapitata MFLU 21-0272 OQ172099 OQ172084 OQ172056 OQ459716 OQ459745 OQ459790 Xiao et al. (2023)
Pleurocordyceps parvicapitata MFLU 21-0273 OQ172100 OQ172085 OQ172057 OQ459717 OQ459746 OQ459791 Xiao et al. (2023)
Pleurocordyceps parvicapitata MFLU 21-0270 OQ172105 OQ172082 OQ172054 OQ459722 OQ459751 OQ459796 Xiao et al. (2023)
Pleurocordyceps parvicapitata MFLU 21-0271T OQ172106 OQ172083 OQ172055 OQ459723 OQ459752 OQ459797 Xiao et al. (2019)
Pleurocordyceps parvicapitata HKAS 131924 PP129615 OR878368 OR835990 OR880682 OR880686 This study
Pleurocordyceps parvicapitata KUNCC23-16075 PP129616 OR878369 OR835991 OR880687 This study
Pleurocordyceps parvicapitata HKAS 131925 OR878366 OR828241 OR880680 OR829677 OR880684 This study
Pleurocordyceps phaothaiensis BCC84551 MF959731 MF959735 MF959739 MF959743 Crous et al. (2017a)
Pleurocordyceps phaothaiensis BCC84552 MF959732 MF959736 MF959740 MF959744 Crous et al. (2017a)
Pleurocordyceps ramosopulvinatus SU-65 DQ118742 DQ118753 DQ127244 Chaverri et al. (2005)
Pleurocordyceps ramosopulvinatus EFCC 5566 KF049658 KF049627 KF049682 KF049645 Kepler et al. (2013)
Pleurocordyceps ramosopulvinatus AB027326 AB027372 Nikoh and Fukatsu (2000)
Pleurocordyceps sinensis CN 80-2 HQ832887 HQ832884 HQ832886 HQ832890 HQ832888 HQ832889 Wang et al. (2012)
Pleurocordyceps sinensis GACP 20-2304 OQ172107 OQ172074 OQ172044 OQ459724 OQ459798 Xiao et al. (2023)
Pleurocordyceps sinensis GACP 20-2305 OQ172108 OQ172075 OQ172045 OQ459725 OQ459753 OQ459799 Xiao et al. (2023)
Pleurocordyceps sinensis MFLU 21-0267 OQ172121 OQ172081 OQ172051 OQ459741 OQ459767 OQ459813 Xiao et al. (2023)
Pleurocordyceps sinensis MFLU 21-0269 OQ172122 OQ172080 OQ172050 OQ459742 OQ459768 OQ459814 Xiao et al. (2023)
Pleurocordyceps sinensis GACP 19-2301 OQ172124 OQ172078 OQ172053 OQ459744 OQ459816 Xiao et al. (2023)
Pleurocordyceps sinensis GZU 20-0865 OQ172096 OQ172071 OQ172043 OQ459713 Xiao et al. (2023)
Pleurocordyceps sinensis HMAS 43720T NR_119928 NG_042573 Wang et al. (2012)
Pleurocordyceps sinensis CGMCC 3.19069 MH454346 MH459160 Sun et al. (2019)
Pleurocordyceps sinensis HQ918290 Zhu et al. (2010)
Pleurocordyceps sp. JB07.08.16_08 KF049616 KF049662 KF049635 KF049690 KF049652 KF049672 Kepler et al. (2013)
Pleurocordyceps sp. JB07.08.17_07b KF049617 KF049691 KF049653 KF049673 Kepler et al. (2013)
Pleurocordyceps sp. BCC 2637 KF049619 KF049663 KF049637 KF049693 KF049675 Kepler et al. (2013)
Pleurocordyceps sp. GIMCC 3.570 JX006097 JX006099 JX006098 JX006100 JX006101 Wang et al. (2020)
Pleurocordyceps sp. NBRC 109990 AB925968 Wang et al. (2020)
Pleurocordyceps sp. NBRC 110224 AB925931 AB925969 Unpublished
Pleurocordyceps sp. NBRC 109987 AB925947 AB925983 Unpublished
Pleurocordyceps sp. NBRC 109988 AB925948 AB925984 Unpublished
Pleurocordyceps sp. HM135166 HM135164 HM135165 Wang et al. (2020)
Pleurocordyceps sp. NBRC 110223 AB925930 Unpublished
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 YHH PY1006T KF977849 KF977851 KF977853 KF977855 Wang et al. (2015a, b)
Pleurocordyceps yunnanensis HAKS 131922 PP129614 OR828244 OR880681 OR829680 This study
Pleurocordyceps yunnanensis YHC PY1005 KF977848 KF977850 KF977852 KF977854 Wang et al. (2015a, b)
Polycephalomyces albiramus GACP 21-XS08T OQ172115 OQ172092 OQ172037 OQ459735 OQ459761 OQ459807 Xiao et al. (2023)
Polycephalomyces albiramus GACPCC 21-XS08T OQ172116 OQ172093 OQ172038 OQ459736 OQ459762 OQ459808 Xiao et al. (2023)
Polycephalomyces formosus NBRC 100686 MN586821 MN586830 MN586839 MN598054 MN598045 MN598061 Wang et al. (2020)
Polycephalomyces formosus NBRC 100687 MN586822 MN586831 MN586840 MN598055 MN598046 MN598062 Wang et al. (2020)
Polycephalomyces formosus NBRC 103843 MN586823 MN586832 MN586841 MN598056 MN598047 MN598063 Wang et al. (2020)
Polycephalomyces formosus NBRC 109993T MN586824 MN586833 MN586842 MN598057 MN598048 MN598064 Wang et al. (2021)
Polycephalomyces formosus NBRC 109994 MN586825 MN586834 MN586843 MN598058 MN598049 MN598065 Wang et al. (2020)
Polycephalomyces formosus GACP 21-WFKQ03 OQ172113 OQ172094 OQ172039 Xiao et al. (2023)
Polycephalomyces formosus GACP 21-WFKQ04 OQ172114 OQ172095 OQ172040 Xiao et al. (2023)
Polycephalomyces ramosus NBRC 101760 MN586818 MN586827 MN586836 MN598051 MN598042 MN598060 Wang et al. (2020)
Polycephalomyces ramosus NBRC 109984 MN586819 MN586828 MN586837 MN598052 MN598043 Wang et al. (2020)
Polycephalomyces ramosus NBRC 109985 MN586820 MN586829 MN586838 MN598053 MN598044 Wang et al. (2020)
Polycephalomyces ramosus MFLU 18-0162T MK863043 MK863250 MK863050 MK860188 Unpublished
Polycephalomyces ramosus NBRC 109983 AB925946 AB925982 Unpublished
Polycephalomyces ramosus RUTPP AY259543 Bischof et al. (2003)
Polycephalomyces ramosus RCEF 6016 KC782530 Crous et al. (2017a)
Polycephalomyces tengchongensis HKAS 131923T PP129612 OR878365 OR828240 OR829676 OR880685 This study
Polycephalomyces tomentosus BL 4 KF049623 KF049666 KF049641 KF049697 KF049656 KF049678 Kepler et al. (2013)
Tolypocladium ophioglossoides NBRC 100998 JN941735 JN943319 JN941406 AB968602 JN992469 AB968563 Ban et al. (2015)
Tolypocladium ophioglossoides NBRC 106330 JN941734 JN943321 JN941407 AB968603 JN992468 AB968564 Ban et al. (2015)

Results

Phylogenetic analyses

The phylogenetic analysis was constructed using sequence data from six loci, representing 112 Polycephalomycetaceae taxa. The alignment comprised 5142 base pair (bp) characters, including gaps (1026 bp for SSU, 602 bp for ITS, 845 bp for LSU, 901 bp for tef-1α, 711 bp for rpb1, and 1057 bp for rpb2). Of these, 3648 characters were constant, 1310 variable characters parsimony-uninformative and 1936 characters parsimony-informative. The likelihood of the best-scoring ML tree was -26079.662.

In the phylogenetic analyses (Fig. 1), two strains of Tolypocladium ophioglossoides (NBRC 100998 and NBRC 106330) were used as outgroup taxa. In the multi-locus phylogenetic tree (Fig. 1), our specimens were distributed across four clades, representing one new genus, one new species and three known species. The strain of Paradingleyomyces lepidopterorum (HKAS 131926, HKAS 131927 and HKAS 131921) formed a distinct clade, positioned between Dingleyomyces and Perennicordyceps, with maximum statistical support (MLBS = 100%, BIPP = 1.00). Polycephalomyces tengchongensis (HKAS 131923) branches off from Polycephalomyces formosus with significant support (MLBS = 100%, BIPP = 1.00). Pleurocordyceps yunnanensis (HKAS 131922) groups with Pleurocordyceps yunnanensis (YHH PY1006 and YHC PY1005) with strong support (MLBS = 84%, BIPP = 0.99). Pleurocordyceps parvicapitata (HKAS 131924), along with its isolate KUNCC23-16075 and the isolate KUNCC23-16074 (which was isolated from the sclerotium of specimen HKAS 131925) clusters with Pleurocordyceps parvicapitata with adequate support (MLBS = 90%, BIPP = 1.00). Perennicordyceps elaphomyceticola (HKAS 131925), which represents the host of Pleurocordyceps parvicapitata phylogenetically clusters with Perennicordyceps elaphomyceticola (MFLU 21-0262, MFLU 21-0263, MFLU 21-0264, MFLU 21-0266, and NTUCC 17-022) with strong support (MLBS = 100%, BIPP = 1.00).

Figure 1. 

Phylogenetic tree of Polycephalomycetaceae based on a concatenated data matrix of SSU, ITS, LSU, tef-1α, rpb1, and rpb2. Bootstrap values greater than or equal to 75% and Bayesian posterior probabilities greater than or equal to 0.80 are shown at the respective nodes. Newly added taxa from this study are highlighted in red.

Taxonomy

Paradingleyomyces Y. Wang tris & T. C. Wen, gen. nov.

Etymology

Morphologically resembling the genus Dingleyomyces.

Type species

Paradingleyomyces lepidopterorum Y. Wang tris & T. C. Wen, sp. nov.

Description

Parasitic on Ophiocordyceps cf. cochlidiicola. Sexual morph: Stroma absent. Perithecia forming from white subiculum covering stromata of Ophiocordyceps cf. cochlidiicola, superficial, scattered, brown, ovoid or ellipsoidal. Asci cylindrical with a thickened cap, attenuated toward the base. Ascospores filiform, hyaline, disarticulating into many cylindrical secondary ascospores at maturity. Secondary ascospores cylindrical, aseptate, smooth-walled, with truncated ends. Asexual morph: Undetermined.

Notes

Both Paradingleyomyces and Dingleyomyces are monotypic genera and share similar morphological characteristics, including the formation of superficial perithecia on a white subiculum, cylindrical asci with thickened caps, and filiform ascospores that disarticulate into cylindrical secondary ascospores. Additionally, the type species of both genera occur as hyperparasites on Ophiocordyceps species (Johnston and Park 2023). However, multigene phylogenetic analysis revealed that these two genera exhibit a paraphyletic relationship, indicating they are not congeneric. Paradingleyomyces can be easily distinguished from Perennicordyceps by its reduced stromata, whereas Perennicordyceps features cylindrical to clavate, branched stromata with prominent rhizomorphs immersed in the substrate, and perithecia forming from the middle to upper parts of the stromata (Ban et al. 2009; Xiao et al. 2019, 2023).

Paradingleyomyces lepidopterorum Y. Wang tris & T. C. Wen, sp. nov.

Fig. 2

Etymology

This epithet is named after the order of its primary host: Lepidoptera.

Description

Parasitic on Ophiocordyceps cf. cochlidiicola. Stromata of host fungus are 55–180 mm in length, 1–3 mm in width, multiple, unbranched, brown at base becoming off-white toward the apex, fibrous, narrowly cylindrical to filiform. Sexual morph: Subiculum white, cottony, covering the stromata of host fungus. Perithecia 240–690 × 110–360 μm (x̄ = 430 × 228 μm, n = 25), emerging from subiculum, superficial, scattered or dense, flesh-colored, ovoid or ellipsoidal. Asci 150–400 × 3–8 μm (x̄ = 289 × 5 μm, n = 30), cylindrical, hyaline, with an apical cap. Apical cap 3–5 × 1–4 μm (x̄ = 3.8 × 2.3 μm, n = 40), hemispherical. Ascospores filiform, multiseptate, breaking into many secondary ascospores at maturity. Secondary ascospores 2–4 × 0.5–1 μm (x̄ = 2.5 × 0.9 μm, n = 50), hyaline, aseptate, smooth-walled, cylindrical with truncated ends. Asexual morph: Undetermined.

Distribution

China: Yunnan Province.

Material examined

Holotype : China • Yunnan Province, Tengchong County, Houqiao Town; 5 Nov. 2022; Collected by Yi Wang; Parasitic on the stromata of Perennicordyceps cf. elaphomyceticola; GYY543H (HKAS 131926) • Paratypes: ibid; GYY543Z (HKAS131927), TC327 (HKAS 131921).

Notes

Paradingleyomyces lepidopterorum lives as a hyperparasite on the remnant stromata of Ophiocordyceps cf. cochlidiicola. The aging stromata of the host fungus become covered with the perithecia of the hyperparasitic fungus, which closely resemble those of the host. However, the key distinguishing feature is that the hyperparasitic perithecia are flesh-colored and grow on a white subiculum, whereas the host’s perithecia are dark brown and directly connected to the stroma (Fig. 2D). Paradingleyomyces lepidopterorum and Dingleyomyces lloydii are morphologically very similar, but they can be easily distinguished from Perennicordyceps species by the presence of a white subiculum from which the perithecia arise (Table 2). In contrast, Perennicordyceps is characterized by cylindrical to clavate, branching stromata with the host and rhizomorphs embedded in the substrate. Dingleyomyces lloydii produce crown-like perithecia on the stromata of Ophiocordyceps hauturu and O. robertsii, while the perithecia of Pa. lepidopterorum sporadically form on the stromata of O. cf. cochlidiicola.

Figure 2. 

Paradingleyomyces lepidopterorum (HKAS 131926, holotype) A habitat B stromata growing from the host C host D perithecia of Pa. lepidopterorum (white arrow) and Ophiocordyceps cf. cochlidiicola (black arrow) E–G perithecia forming on white subiculum H vertical section of perithecium. I Peridium J–L asci M, N apical cap of asci O–Q secondary ascospores. Scale bars: 1 mm (E–G); 500 μm (H); 100 μm (I); 50 μm (J, K); 25 μm (L); 20 μm (M, N); 5 μm (O–Q).

Table 2.

Morphological comparison between sexual species in Paradingleyomyces, Perennicordyceps, and Dingleyomyces.

Species Host Stromata (mm) Perithecia (µm) Asci (µm) Apical cap (µm) Secondary ascospores (µm) References
Dingleyomyces lloydii Ophiocordyceps hauturu, Ophiocordyceps robertsii Reduced to white subiculum, flat, thin, irregular plates, often obscured by the perithecia, white or yellowish 300–950 × 300–650, superficial, ovoid, growing in small groups on white subiculum 200–450 × 6–10 2–3 diameter, 4 thickness 1.5–3 × 1–1.5 Dingley (1953); Mains (1958); Johnston and Park. (2023)
Perennicordyceps elaphomyceticola Elaphomyces sp. 20–100 × 0.1–0.5, cylindrical, the colours vary from dark brown, titian red, brownish orange, yellow to pale 430–600 × 255–300, superficial, ovoid to ellipsoid, yellow when mature, pale when immature 365–420 × 5.0–7.6 2–3.5 × 3.3–5.2 1.5–3.2 × 1.4–2.3 Xiao et al. (2023)
Pe. cuboidea Beetle larva or other Cordyceps 32–181 × 3–74, cylindrical, ochre yellow 400–500 × 250–330, superficial, lemon-shaped, glabrate 250–570 in length 3–5 in thickness 1.5–3 × 1–1.5 Ban et al. (2009)
Pe. prolifica Cicada nymph 70.9–140.0× 0.8–2.2, thin cylindrical, brown 320–530 × 200–340, superficial, ovoid or ellipsoidal, grayish brown 430–650 in length 3–5 in thickness 2–3 × 1–2 Kobayasi and Shimizu (1963)
Pa. lepidopterorum Ophiocordyceps cf. cochlidiicola Reduced to white subiculum 240–690 × 110–360, superficial, ovoid to ellipsoidal, brown 150–400 × 3–8 3–5 × 1–4 2–4 × 0.5–1 This study
Pe. paracuboidea Coleopteran larva 3.2–38.4 × 0.3–1.7, cylindrical 400–600 × 290–400, superficial, lemon-shaped, pale orangish brown 225–400 in length 3–6.3 in thickness 1.3–2.5 × 1–2 Ban et al. (2009)
Pe. ryogamiensis Coleopteran larva 12–13 × 0.5, cylindrical, white, palely darkened, glabrate at the base 320–430 × 200–230, superficial, ovoid 450–610 in length 3.8–5 in thickness 2.5–5 × 1.5–2 Ban et al. (2009)

Polycephalomyces tengchongensis Y. Wang tris & T. C. Wen, sp. nov.

Fig. 3

Etymology

Named after the location where the type specimen was found, Tengchong County, Yunnan Province.

Description

Parasitic on Perennicordyceps cf. elaphomyceticola. Sexual morph: Undetermined. Asexual morph: Synnemata 18.7 mm long, 1–2 mm wide, cylindrical, white, growing in small group on stromata of Perennicordyceps cf. elaphomyceticola. Fertile parts yellowish, with conidial mass forming on middle part of synnemata. Phialides dimorphic. α-phialides 9–20 × 1–2 μm (x̄ = 12.3 × 1.2 μm, n = 45), phialidic, subulate, hyaline, smooth-walled, arranged in a parallel palisade-like layer around the fertile part. α-conidia 1–3 μm (x̄ = 2 μm, n = 45), globose, hyaline.

Figure 3. 

Polycephalomyces tengchongensis (B–I from HKAS 131923, J–R from KUNCC23-16073) A habitat B–F infected Perennicordyceps cf. elaphomyceticola showing synnemata of parasites G, H α-phialides I α-conidia J–M β-phialides N–P β-conidia Q, R reverse and front view of culture on PDA. Scale bars: 5 mm (D–F); 20 μm (G, H); 50 μm (J–L); 10 μm (M); 5 μm (I, N–P).

Culture characters

Colonies on PDA attaining a diam. of 28–31 mm in 14 days at 25 °C, white, leathery, radially striate, reverse dark brown and turns pale outward. β-phialides 18–44 × 1–3 μm (x̄ = 26.7 × 1.2 μm, n = 30), phialidic, lanceolate, smooth-walled. β-conidia 3–7 × 1.5–3 μm (x̄ = 3.9 × 2.2 μm, n = 45), ellipsoidal to broadly fusiform, hyaline, aseptate, smooth-walled.

Material examined

China • Yunnan Province, Tengchong County, Houqiao Town; 5 Nov. 2022; Collected by Yi Wang; Parasitic on the stromata of Perennicordyceps cf. elaphomyceticola; GYY547 (HKAS 131923, ex-holotype living culture: KUNCC23-16073).

Notes

The newly described species Polycephalomyces tengchongensis is closely related to Po. formosus with strong support (MLBS = 100%, MIPP = 1.00, Fig. 1). However, Polycephalomyces tengchongensis is distinct from Po. formosus in several aspects. It parasitizes Perennicordyceps cf. elaphomyceticola and produces synnemata without well-defined stipe and a fertile head but features dimorphic phialides and conidia. In contrast, Po. formosus has stipitate synnemata with a fertile head at the tip and produces only one type of phialides and conidia (Xiao et al. 2023).

A comparison of nucleotide sequences between Po. tengchongensis and the ex-type of strain of Po. formosus (NBRC 109993) revealed 1% differences (6/584 bp) including three gaps in the ITS region, 0.3% (3/774 bp) differences including one gap in the LSU region, 2.3% differences (16/684 bp) including three gaps in the rpb1 region and 1.6% differences (15/891bp) in the rpb2 region. Collectively, the differences both in phenotypic profiles and nucleotides sequences support the establishment of Polycephalomyces tengchongensis as a new species.

Pleurocordyceps parvicapitata Y.P. Xiao, T.C. Wen & K.D. Hyde, in Xiao et al. Fungal Diversity 120: 50 (2023)

Figs 4, 5

Description

Parasitic on Elaphomyces sp. (Fig. 4). The host 6–10 mm in diam., dark brown or brown, spherical, hard, and rough on the surface. Sexual morph: Stromata 18–21 mm long, 1–2 mm wide, brown, multiple, fibrous. Stipe 8–15 mm long, 0.5–1 mm wide, brown, cylindrical, terminally or laterally carrying fertile cushions. Fertile cushions 0.5–1 mm in height, 1–2 mm in width, pale yellow to yellow, hemispherical. Perithecia 160–530 × 100–305 µm (x̄ = 306 × 179 µm, n = 20), immersed, crowded, ovoid to obpyriform, ostiolate. Peridium 15–40 µm (x̄ = 25 µm, n = 20) wide, three-layered, comprised of hyaline to pale yellow cells of textura intricate at outermost layer to textura angularis at middle layer to textura prismatica at inner layer. Asci 190–380 × 3–5 µm (x̄ = 252 × 3.9 µm, n = 50), cylindrical, with thickened apex. Apical cap 1–2 × 2.5–4 μm (x̄ = 1.7 × 3.4 µm, n = 60), hyaline. Ascospores filiform, multiseptate, hyaline, breaking into many secondary ascospores at maturity. Secondary ascospores 2–8 × 0.5–1 µm (x̄ = 5.1 × 0.9 µm, n = 50), cylindrical, aseptate, straight, smooth-walled. Asexual morph: Synnemata cylindrical, off-white, gregarious, unbranched, occurring in close proximity to the fertile cushions. β-phialides up to 16 µm in length, 2 µm in width, cylindrical, attenuate toward the apex, phialidic, hyaline, smooth-walled. β-conidia 3–5 × 1–2 µm (x̄ = 3.8 × 1.7 µm, n = 20), fusiform, hyaline, aseptate. Additionally, Pleurocordyceps parvicapitata parasitic on Perennicordyceps elaphomyceticola was found in proximity to the one on Elaphomyces sp. (Fig. 5). Sexual morph: Stromata not observed. Fertile cushion 0.5–1 mm long, 1–2 mm wide, directly growing on stromata of Pe. elaphomyceticola, pale yellow to yellow, surface wrinkle, rough due to the protruding perithecia. Perithecia 440–560 ×115–250 μm (x̄ = 505 × 170 µm, n = 15) ovoid to obpyriform, immersed, gregarious. Peridium 15–42 µm (x̄ = 25 µm, n = 20) wide, three-layered, comprised of hyaline to pale yellow cells of textura intricate at outermost layer to textura angularis at middle layer to textura prismatica at inner layer. Asci and ascospores were not observed due to the specimen being immature.

Figure 4. 

Pleurocordyceps parvicapitata (B–R from HKAS 131924 S–Z from KUNCC23-16075) A habitat B, C stromata emerging from host D host (Elaphomyces sp.) E fertile cushions on stromata F enlargement of fertile cushion G cross-section through fertile cushion H perithecium I peridium J, P synnemata on stromata K asci L part of asci M ascus cap N, O secondary ascospores Q, V, W β-phialides S front and reverse view of culture on PDA T synnemata on culture U, X α-phialides Y α-conidia Z β-conidia. Scale Bars: 5 mm (E); 1 mm (D, F); 500 µm (G, J, P); 250 µm (H); 100 µm (I); 50 µm (K, L); 20 µm (M, N, V, U, W); 10 µm (X); 5 µm (O, Q, R, Y, Z).

Figure 5. 

Pleurocordyceps parvicapitata (HKAS 131925) A habitat B, C, E host (Perennicordyceps elaphomyceticola) D, F, G fertile cushions growing on stromata of Pe. elaphomyceticola H cross-section through fertile cushion I perithecium J peridium L reverse and front view of culture on PDA after incubation for 14 days M, N front view of culture on PDA after incubation for 30 days O synnemata P conidiophores Q phialides R conidia. Scale bars: 1 mm (E–G); 500 µm (H, I, O); 20 µm (J, P, Q); 5 µm (R).

Culture characters

Colonies isolated from Elaphomyces sp. and Perennicordyceps elaphomyceticola present similar characteristics. Colonies on PDA attaining 41–45 mm in diam. after incubation at 25 °C for 14 days, white, leathery, reverse grayish yellow. Synnemata emerging from margin of colony in annular distributions, 1–3 mm long, 1–2.5 mm wide, white, erected, apically branched. Conidial mass gathers at middle part or tip of synnemata, pale yellow, waterish. Phialides two-typed. α-phialides 10–28 × 1–2 µm (x̄ = 15.7 × 1.5 µm, n = 50), hyaline, smooth-walled, subculate, caespitose, palisade-like. α-conidia 1–2 µm (x̄ = 1.7 µm, n = 45), one-celled, hyaline, smooth-walled, globose. β-phialides 6–8 × 0.5–1 µm (x̄ = 7.9 × 1.1 µm, n = 20), arising from hypha, solitary, lanceolate, hyaline, smooth-walled. β-conidia 2–6 × 1–2 µm (x̄ = 3.8 × 0.8 μm, n = 35), fusiform, hyaline, aseptate, smooth-walled, asymmetrical.

Material examined

China • Yunnan Province, Tengchong County, Houqiao Town; 5 Nov. 2022; Collected by Yi Wang; Parasitic on Elaphomyces sp. buried in soil; GYY546 (HKAS 131924, living culture: KUNCC23-16075) • ibid; Parasitic on Perennicordyceps elaphomyceticola; 5 Nov. 2022; Collected by Yi Wang; GYY553 (HKAS 131925, living culture: KUNCC23-16074).

Notes

Pleurocordyceps parvicapitata, parasitic on Elaphomyces sp. and Perennicordyceps elaphomyceticola, was originally described by Xiao et al. (2023) based on specimens collected from Dadugang County, Xishuangbanna, Yunnan Province, China. The specimen associated with Elaphomyces sp. produces pale yellow to yellow, wrinkled fertile cushions that are laterally or terminally attached to stromata, along with cylindrical asci, filiform, disarticulating ascospores and cylindrical, smooth-walled secondary ascospores. In this study, we collected a specimen displaying the typical characteristics of Pl. parvicapitata from Tengchong County, Yunnan Province. Importantly, Xiao et al. (2023) described Pl. parvicapitata as having one type of phialides and conidia from dry specimen. In contrast, we examined the asexual morph from both our specimens and its pure culture, observing dimorphic phialides and conidia. Additionally, the specimen associated with Perennicordyceps elaphomyceticola was previously known only from its asexual morphs (Xiao et al. 2023), where the species was described as having pulvinate, yellowish conidiomata with one-type of phialides and conidia on the stromata of Pe. elaphomyceticola. In this study, we collected a sexual specimen from Tengchong County, Yunnan Province and its fertile cushion was similar to Pl. parvicapitata found on Elaphomyces sp. (Xiao et al. 2023). This is the first report of the sexual morph of Pl. parvicapitata on Pe. elaphomyceticola, which differs from previously described sexual morphs in that it directly forms a fertile cushion on the substrate. We have also supplemented this species with a pure culture which can be used for further research. These findings provide deeper insights into the morphological traits of Pl. parvicapitata.

Pleurocordyceps yunnanensis (Hong Yu bis, Y.B. Wang & Y.D. Dai) Y.H. Wang, S. Ban, W.J. Wang, Yi Li, Ke Wang, P.M. Kirk & Y.J. Yao, in Wang et al. Journal of Systematics and Evolution 59(5): 1076 (2021)

Fig. 6

Description

Parasitic on Ophiocordyceps nutans (Ophiocordycipitaceae, Hypocreales) (Fig. 6). Sexual morph: Stromata 12–25 mm long, 0.5–1 mm wide, fibrous, brown, multiple, unbranched. Stipes 5–11 mm long, ca. 0.5 mm wide, brown to pale brown. Fertile head 1–2.5 mm long, 0.7–1.3 mm wide, yellowish to yellow, capitate, rough. Perithecia 160–390 × 55–170 μm (x̄ = 269 × 115 µm, n = 20), immersed, crowded, ovoid to obpyriform, ostiolate yellow, thick-walled. Peridium 14–46 µm (x̄ = 32 µm, n = 25) wide, three-layered, comprised of hyaline to yellowish cells of textura prismatica at outer layer to textura angularis at middle layer to textura porrecta at inner layer. Asci 95–235 × 3–6 µm (x̄ = 172 × 5 µm, n = 40), 8-spored, with thickened cap. Ascospores filiform, hyaline, multiseptate, disarticulating into many secondary ascospores at maturity. Secondary ascospores 2.5–5 × 1–2 µm (x̄ = 3.9 × 1.3 µm, n = 40), cylindrical, aseptate, hyaline, smooth-walled. Asexual morphs: see Wang et al. (2015a).

Figure 6. 

Pleurocordyceps yunnanensis (HAKS 131922) A habitat B–D stromata of Pl. yunnanensis growing from the host (Ophiocordyceps nutans) E fertile head F cross-section through fertile head G perithecium H peridium I–K asci L, M part of asci N part of ascospore O, P secondary ascospores. Scale bars: 5 mm (C, D); 1 mm (E); 100 µm (F, G); 50 µm (H–K); 20 µm (L, M); 5 µm (N–P).

Material examined

China • Yunnan Province, Kunming City, the Wild Duck Lake Forest Park; 17 Sep. 2023; Collected by Yi Wang; Parasitic on Ophiocordyceps nutans; YYH13 (HAKS 131922).

Notes

The asexual morph of Polycephalomyces yunnanensis was first described by Wang et al. (2015a) from Ophiocordyceps nutans in Wild Duck Lake Forest Park, Kunming, Yunnan Province. This species was later transferred to Pleurocordyceps by Wang et al. (2021) based on molecular phylogenetic analyses. In this study, a sexual polycephalomyces-like fungus growing on O. nutans was obtained from the same location as the type specimen (Fig. 6). Phylogenetic analysis revealed that it groups with strains of Pleurocordyceps yunnanensis with strong support (Fig. 1). Therefore, we introduce our specimen as the new sexual morph of Pl. yunnanensis.

Discussion

Polycephalomycetaceae was introduced by Xiao et al. (2023) to encompass the genera Pleurocordyceps, Perennicordyceps, and Polycephalomyces. The monophyletic nature of these three genera has been confirmed through numerous phylogenetic studies (Tian et al. 2010; Wang et al. 2012; Kepler et al. 2013; Wang et al.2015a; Zhong et al. 2016; Crous et al. 2017a; Sobczak etal. 2017; Poinar and Vega 2020; Xiao et al. 2018, 2023). Johnston and Park (2023) introduced a new genus, Dingleyomyces, into Polycephalomycetaceae. Dingleyomyces is a monotypic genus, and typified by Dingleyomyces lloydii, a species that is hyperparasitic on Ophiocordyceps hauturu from New Zealand. Dingleyomyces lloydii was placed in a distant clade branching off from Perennicordyceps (Johnston and Park 2023). In this study, we introduce a new genus, Paradingleyomyces to accommodate Pa. lepidopterorum which forms a distinct clade nested between Perennicordyceps and Dingleyomyces (Fig. 1).

Perennicordyceps currently comprises six species, four identified based on their sexual morphology and two based on their asexual morphology. We have compared the sexual characteristics of Pa. lepidopterorum with the four sexual species of Perennicordyceps, as depicted in Fig. 7. Several distinctions between Paradingleyomyces and Perennicordyceps are observed: 1) Pa. lepidopterorum is characterized by the absence of stromata, while Perennicordyceps species exhibit branched, cylindrical to clavate, rhizomorphic stromata; 2) The host of Pa. lepidopterorum is Ophiocordyceps cf. cochlidiicola, whereas Perennicordyceps parasitize a broader range of host, including insect and fungi; 3) The perithecia of Pa. lepidopterorum form on a white subiculum and are distributed over the entire stromata of the host fungus, while in Perennicordyceps species, perithecia are densely formed from the middle to the upper part of the stromata; 4) The length-to-width ratio of secondary ascospores in Pa. lepidopterorum is 2.8: 1, which is greater than that observed in Perennicordyceps species. Consequently, we introduced Paradingleyomyces as a distinct genus rather than categorizing it within Perennicordyceps. Although Dingleyomyces has shares the poorly developed stromata connecting crowed perithecia to the stromata of Ophiocordyceps hauturu and Ophiocordyceps robertsii, giving a similar appearance to Pa. lepidopterorum, multigene phylogeny revealed a paraphyletic relationship between Dingleyomyces and Paradingleyomyces. Therefore, the establishment of Paradingleyomyces is well-supported by both morphological observations and phylogenetic analysis. The asexual morph of Paradingleyomyces is currently unknown, and future efforts should focus on exploring more hidden species within this genus.

Figure 7. 

Morphological comparison of Paradingleyomyces, Dingleyomyces and Perennicordyceps. In Perennicordyceps, the dotted line below indicates burial in soil or woods. Pe. cuboidea, Pe. prolifica, Pe. paracuboidea and Pe. ryogamiensis were redrawn from Ban et al. (2009) and Pe. elaphomyceticola was redrawn from Xiao et al. (2023). Pa. lepidopterorum is a newly described species in this study. D. lloydii was redrawn from Johnston and Park (2023).

Polycephalomyces was initially classified under Hypocreales incertae sedis (Kepler et al. 2013; Matočec et al. 2014). Up to 25 species were assigned to Polycephalomyces, but later some were transferred to Pleurocordyceps and Perennicordyceps, remaining eight species: Po. albiramus, Po. formosus, Po. ramosus, Po. tomentosus, Po. baltica, Po. cylindrosporus, Po. ditmarii, and Po. paludosus (Xiao et al. 2023). However, the phylogenetic relationships of the latter four species remain unclear due to a lack of molecular data. Notably, Po. ramosus and Po. tomentosus group with species of Pleurocordyceps in the study of Xiao et al. (2023) and this phylogenetic relationship is also observed in our study. Thus, the taxonomic status of Po. ramosus and Po. tomentosus remains questionable. In this study, we introduce a new species, Polycephalomyces tengchongensis, which is parasitic on Perennicordyceps cf. elaphomyceticola from Tengchong County, Yunnan Province, China. This new species is distinguished by a unique combination of features, including its host association, synnemata lacking a stipe and fertile head, and the presence of dimorphic phialides and conidia (see Table 3). The finding of Po. tengchongensis adds to the morphological diversity within the genus Polycephalomyces.

Table 3.

Distinguishing characteristics between Po. tengchongensis and other Polycephalomyces species.

Species Host Synnemata Phialides (µm) Conidia (µm) Reference
Po. tengchongensis Perennicordyceps cf. elaphomyceticola (Hypocreales, Polycephalomycetaceae) Non-stipitate, without fertile head Two-type, α-phialides 9–20 × 1–2, subulate; β-phialides 18–44 × 1–3, lanceolate Two-type, α-conidia 1–3, globose; β-conidia 3–7 × 1.5–3, ellipsoidal to broadly fusiform This study
Po. albiramus Gryllotalpa sp. (Orthoptera) Stipitate, without fertile head One-type, 12.8–18.3 × 1–2.2, narrowly subulate, awl-shaped One-type 2.1–3.2 × 0.9–1.2, cylindrical to obovoid or subglobose Xiao et al. (2023)
Po. baltica Nymph or short-winged female bark louse Stipitate, with fertile head One-type, 3–4 long, flask-shaped One-type, 3–4, globose Poinar and Vega (2020)
Po. cylindrosporus Coleoptera, Formicidae and Pentatomidae Stipitate, with fertile head One-type, 7–25 long One-type, 2.5–4, cylindrical to bacilliform Matočec et al. (2014)
Po. ditmarii Paravespula vulgaris (Wasp), fly Stipitate, with fertile head One-type, 20–37 × 1.5–2.5, elongate, cylindrical, attenuating at the apex One-type, 2.2–3 × 1.3–1.6, globose to subglobose to clavate Van Vooren and Audibert (2005), Xiao et al. (2023)
Po. formosus Coleopteran larvae or Ophiocordyceps barnesii Stipitate, with fertile head One-type, 6–25 × 1–1.2, cylindrical, tapering gradually One-type, 2.5–3.2 × 1–1.2, ellipsoidal or ovoid Kobayasi (1941)
Po. ramosus Lepidopteran larvae or Hirsutella guignardii Stipitate, with fertile head Two-type, α-phialides 7–24 long, 1–2 at basal wide, cylindrical to narrowly lageniform; β-phialides 6–27 long, 2–3.5 at basal wide, 0.5–1 at neck width, narrowly lageniform or subulate Two-type, α-conidia 2.4–3.2 × 1.6–2.4, ovoid; β-conidia 3.2–4 × 1.6–2, fusiform Seifert (1985), Bischof et al. (2003)
Po. paludosus Lepidopteran larva Stipitate, with fertile head One-type, 2–20 long, 1–1.5 at basal wide, subulate One-type, 8–2.5 × 1.1–1.3, obovoid Mains (1948)
Po. tomentosus Myxomycetes Three-type, globose or ellipsoidal or cylindrical Seifert (1985)

Pleurocordyceps was introduced by Wang et al. (2021) to accommodate ten species: Pleurocordyceps agarica, Pl. aurantiacus, Pl. lianzhouensis, Pl. marginaliradians, Pl. nipponica, Pl. onorei, Pl. phaothaiensis, Pl. ramosopulvinatus, Pl. sinensis, and Pl. yunnanensis, based on phylogenetic analysis. Wei et al. (2022) added a new species Pleurocordyceps ophiocordycipiticola which parasitizes Ophiocordyceps cylindrospora in Thailand. Xiao et al. (2023) introduced five additional species to this genus, including Pl. heilongtanensis, Pl. lanceolata, Pl. nutantis, Pl. parvicapitata and Pl. vitellina. Currently, Pleurocordyceps comprises 16 species, all of which have been verified by molecular phylogeny. The sexual morph of Pleurocordyceps is characterized by stipitate, fibrous stromata that produce pale yellow to yellow fertile cushion either laterally or terminally, with immersed ostiolate perithecia, cylindrical asci, filiform disarticulating ascospores and cylindrical secondary ascospores. The asexual morph is characterized by stipitate, non-stipitate, or pulvinate synnemata, with or without fertile heads, generally displaying dimorphic phialides and conidia. Sexual morphs have been identified in six species including Pl. marginaliradians (Xiao et al. 2018), Pl. nipponica, Pl. onorei (Crous et al. 2017a), Pl. parvicapitata (Xiao et al. 2023), Pl. phaothaiensis (Crous et al. 2017b) and Pl. ramosopulvinata (Wang et al. 2021). The remaining 10 species of Pleurocordyceps have been described based on their asexual morphs. In this study, we report the sexual morph of Pl. yunnanensis from Ophiocordyceps nutans for the first time, collected from the same location as the type specimen. Ecologically, Pleurocordyceps species are particularly prone to infecting Ophiocordyceps or Perennicordyceps species, as well as their insect or fungal hosts. For instance, Pl. parvicapitata is known to infect Perennicordyceps elaphomyceticola and its host Elaphomyces sp. at the same region (Xiao et al. 2023). In this study, we once again obtained Pl. parvicapitata which infects both Pe. elaphomyceticola and its host Elaphomyces sp. from Tengchong County, Yunnan Province. This finding indicates that Pl. parvicapitata may be specific to Pe. elaphomyceticola and Elaphomyces sp. Additionally, we are the first to isolate and observe dimorphic phialides and conidia in Pl. parvicapitata, while Xiao et al. (2023) reported only one type of phialides and conidia from dried specimen. Therefore, the presence of dimorphs phialides and conidia should not be considered a reliable feature for species demarcation within Pleurocordyceps.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This research was funded by the Science and Technology Foundation of Guizhou Province (No. [2019] 2451-3), the National Natural Science Foundation of China (No. 31760014), and Thailand Science Research and Innovation (TSRl) grant No.672A01014 from the Fundamental Fund for Basic Research, National Research Council of Thailand.

Author contributions

Investigation: YZ, GYW, XZ. Resources: XCP, XSH. Writing – original draft: TCW, YW. Writing – review and editing: DPW, JCK, CRL, PC, RCL, ZZL. All authors have read and agreed to the published version of the manuscript.

Author ORCIDs

Yi Wang https://orcid.org/0009-0006-5412-7893

De-Ping Wei https://orcid.org/0000-0002-3740-0142

Xing-Can Peng https://orcid.org/0000-0002-7271-7639

Ji-Chuan Kang https://orcid.org/0000-0002-6294-5793

Xian Zhang https://orcid.org/0009-0008-0919-4303

Putarak Chomnunti https://orcid.org/0000-0003-2989-1735

Ting-Chi Wen https://orcid.org/0000-0003-1744-5869

Data availability

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

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