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Research Article
Multigene phylogeny and morphology reveal Ophiocordyceps hydrangea sp. nov. and Ophiocordyceps bidoupensis sp. nov. (Ophiocordycipitaceae)
expand article infoWeiqiu Zou‡, Dexiang Tang‡, Zhihong Xu‡, Ou Huang‡, Yuanbing Wang‡, Ngoc-Lan Tran§, Hong Yu‡
‡ Yunnan University, Kunming, China
§ Institute of Regional Research and Development, Ministry of Science and Technology, Hanoi, Vietnam

Corresponding author: Hong Yu ( hongyu@ynu.edu.cn )

Academic editor: Cecile Gueidan
© 2022 Weiqiu Zou, Dexiang Tang, Zhihong Xu, Ou Huang, Yuanbing Wang, Ngoc-Lan Tran, 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.
Citation: Zou W, Tang D, Xu Z, Huang O, Wang Y, Tran N-L, Yu H (2022) Multigene phylogeny and morphology reveal Ophiocordyceps hydrangea sp. nov. and Ophiocordyceps bidoupensis sp. nov. (Ophiocordycipitaceae). MycoKeys 92: 109-130. https://doi.org/10.3897/mycokeys.92.86160
Open Access

Abstract

Ophiocordyceps species have a wide range of insect hosts, from solitary beetle larva to social insects. However, among the species of Ophiocordyceps, only a few attack cicada nymphs. These species are mainly clustered in the Ophiocordyceps sobolifera clade in Ophiocordyceps. A new entomopathogenic fungus parasitic on cicada nymphs, and another fungus parasitic on the larva of Coleoptera, are described in this study. The two new species viz. Ophiocordyceps hydrangea and Ophiocordyceps bidoupensis were introduced based on morphology and multigene phylogenetic evidence. The phylogenetic framework of Ophiocordyceps was reconstructed using a multigene (nrSSU, nr LSU, tef-1α, rpb1, and rpb2) dataset. The phylogenetic analyses results showed that O. hydrangea and O. bidoupensis were statistically well-supported in the O. sobolifera clade, forming two separate subclades from other species of Ophiocordyceps. The distinctiveness of these two new species was strongly supported by both molecular phylogeny and morphology.

Keywords

2 new taxa, entomopathogenic fungi, morphology, phylogenetic analyses

Introduction

Ophiocordyceps G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora is the largest genus in the Ophiocordycipitaceae, comprising approximately 290 species. It was originally established by Petch, with Ophiocordyceps blattae Petch as the type species (Petch 1931). According to the arrangement of the perithecia, the size of asci, ascospores, and secondary ascospores, Ophiocordyceps was transferred to Cordyceps sensu lato by Kobayasi, as a subgenus of Cordyceps s.l. (Kobayasi 1941, 1982). Sung et al. (2007) used five to seven loci combined molecular datasets to revise the Cordyceps and the Clavicipitaceae. The species of Cordyceps and Clavicipitaceae were divided into three families (Cordycipitaceae, Ophiocordycipitaceae, Clavicipitaceae sense stricto) and four genera (Cordyceps sense stricto, Ophiocordyceps, Elaphocordyceps, and Metacordyceps). The research results of Sung et al. (2007) are currently the most widely accepted phylogenetic classification of Cordyceps s.l. In 2015, Ophiocordyceps was divided into O. ravenelii clade, O. unilateralis clade, O. sobolifera clade, and O. sphecocephala clade by Sanjuan et al. With the continuous revision of Ophiocordyceps, it has now been divided into four clades, including the Hirsutella clade, O. sobolifera clade, O. sphecocephala clade, and O. ravenelii clade (Mains 1958; Sung et al. 2007; Quandt et al. 2014; Sanjuan et al 2015; Simmons et al. 2015; Wang et al. 2018). Many phylogenetic classifications for entomopathogenic fungi have been revised in recent studies (Wang et al. 2018; Fan et al. 2021; Wang et al. 2021a, 2021b).

There are fewer species in the O. sobolifera clade than in the Hirsutella clade and the O. sphecocephala clade. The O. sobolifera clade is statistically well-supported in most studies and 11 species have been described in the Index Fungorum (Kobayasi and Shimizu 1963; Hywel-Jones 1995b; Sung et al. 2007, 2011; Luangsa-ard et al. 2008; Hyde et al. 2017; Crous et al. 2018, 2019; Lao et al. 2021; Wang et al. 2021a). Asexual morphs of Ophiocordyceps were reported as Hirsutella Pat., Paraisaria Samson & B.L. Brady, Sorosporella Sorokin, Hymenostilbe Petch and Syngliocladium Petch, etc. (Sung et al. 2007; Quandt et al. 2014). In most species of Ophiocordyceps, their dominant asexual morphs were Hirsutella, the conidiogenous cells basally swollen that taper to a narrow neck, producing a mucilaginous cluster of one or several conidia (Simmons et al. 2015; Wang et al. 2018).

Ophiocordyceps species have a wide range of insect hosts, from solitary beetle larvae to social insects. More than 10 insect orders were attacked, including Hemiptera, Coleoptera, Lepidoptera, Blattaria, Dermaptera, Diptera, Hymenoptera, Isoptera, Megaloptera, and Mantodea (Araújo et al. 2015; Araújo and Hughes 2016, 2019). Entomopathogenic fungi whose hosts are cicada nymphs have attractive stromata. The most typical representative of this group was Cordyceps cicadae (Miquel) Massee (Massee 1895) in Cordycipitaceae, with the stroma like a flower (Sung et al. 2007). However, for species of Ophiocordyceps, with cicada nymph hosts including O. khonkaenensis Tasanathai, Thanakitpipattana & Luangsa-ard (Crous et al. 2019), O. sobolifera (Hill ex Watson) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora (Kobayasi and Shimizu 1963; Sung et al. 2007), and O. longissima (Kobayasi) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora (Kobayasi and Shimizu 1963; Sung et al. 2007, 2011) in O. sobolifera clade, their stromata were typically bright-colored and cylindrical. The hosts of the entomopathogenic fungi within the O. sobolifera clade were divided into two categories. One group with Hemiptera hosts was represented by O. sobolifera. These fungi had a hard texture stroma, which was cylindrical, and deep-colored, and had swollen fertile parts (Kobayasi and Shimizu 1963; Sung et al. 2011; Crous et al. 2019). Another group had Coleoptera hosts that were characterized by hard texture stromata, being cylindrical, bright-colored, and with a sterile apices cone at the top of the stroma (Hywel-Jones 1995b; Luangsa-ard et al. 2008; Crous et al. 2018; Lao et al. 2021; Wang et al. 2021a).

Cordyceps s.l. is globally distributed with the highest species diversity recorded in subtropical and tropical regions (Nguyen and Vo 2005; Ban et al. 2015; Doan et al. 2017; Luangsa-ard et al. 2018), especially in East and Southeast Asia (Sung et al. 2007; Fan et al. 2021; Wang et al. 2021a). To date, more than 800 species of Cordyceps and Ophiocordyceps have been named worldwide, and there are at least 200 species in China (Index Fungorum 2022). Yunnan Province, located in southwest China, has unique geographical and ecological features. Many species of Ophiocordyceps were reported from Yunnan, including O. alboperitheciata H. Yu, Q. Fan & Y.B. Wang (Fan et al. 2021), O. furcatosubulata H. Yu, Y. Wang & Y.B. Wang (Wang et al. 2021a), O. highlandensis Zhu L. Yang & J. Qin (Yang et al. 2015), O. lanpingensis H. Yu & Z.H. Chen (Chen et al. 2013), O. laojunshanensis J.Y. Chen, Y.Q. Cao & D.R. Yang (Chen et al. 2011), O. liangshanensis (M. Zang, D.Q. Liu & R.Y. Hu) H. Yu, Y. Wang, Y.D. Dai, Zhu L. Yang & Y.B. Wang (Wang et al. 2021b), and O. pingbianensis H. Yu, S.Q. Chen & Y.B. Wang (Chen et al. 2021). The unique geographical conditions of Yunnan have resulted in high Cordyceps s.l. species diversity. There is also a high species diversity of Cordyceps s.l. in Southeast Asia, where more than 500 species of entomopathogenic fungi have been reported. Approximately 400 species of entomopathogenic fungi are distributed in Thailand (Sung et al. 2007; Luangsa-ard et al. 2011, 2018; Ban et al. 2015; Tasanathai et al. 2019; Xiao et al. 2019). Vietnam is second to Thailand, in the number of entomopathogenic fungi species, with more than 100 species having been reported such as Moelleriella pumatensis T.T. Nguyen & N.L. Tran (Mongkolsamrit et al. 2011), O. furcatosubulata H. Yu, Y. Wang & Y.B. Wang (Wang et al. 2021a), and O. puluongensis H. Yu, Z.H. Xu, N.L. Tran & Y.B. Wang (Xu et al. 2022). These findings suggested that Vietnam should be abundant in species diversity of Cordyceps s.l. (Mongkolsamrit et al. 2011; Doan et al. 2017; Luyen et al. 2017).

Several studies have evaluated the taxonomy and biology of entomopathogenic fungi, especially species found in China and Southeast Asia. In this study, one unknown species of Ophiocordyeps attacking a cicada nymph was collected from Yunnan Province, Jinghong City, Nabanhe National Nature Reserve, in China. Another unknown species of Ophiocordyeps attacking larvae of Elateridae was collected from Lintong Province, Bidoup Nuiba National Park, in Vietnam. The phylogeny and morphology of these two fungi were determined, and their systematic position was established in Ophiocordycipitaceae. The phylogenetic analyses results showed that the two new species belonged to Ophiocordyceps, and were named Ophiocordyceps hydrangea and Ophiocordyceps bidoupensis based on well-supported morphology and molecular data.

Materials and methods

Sample collection and isolation

The specimens were collected from China and Vietnam, and the collection site information was noted, including altitude, longitude, latitude, and habitat type. Samples were placed in sterilized tubes or plastic bags and boxes, returned to the laboratory, and stored at 4 °C. The specimens were photographed using a Canon 750 D camera (Canon Inc., Tokyo, Japan). The size was measured, and characteristics were recorded including length of the stroma, single or multiple, length and width of stipe clavate and fertile parts, shape, texture, and color. To obtain axenic cultures, the segments were removed from insect bodies, and these segments were placed onto Potato Dextrose Agar (PDA) consisting of peptone and yeast powder (potato 100 g/500 mL, dextrose 10 g/500 mL, agar 10 g/500 mL, yeast powder 5 g/500 mL, peptone 2.5 g/500 mL) plates. The plates were placed in a culture room at 25 °C until isolated into pure cultures. The cultures were saved on a PDA slant (to grow slowly), and stored at 4 °C. All specimens were deposited in the Yunnan Herbal Herbarium (YHH) of Yunnan University. The extypes of the two species were deposited in the Yunnan Fungal Culture Collection (YFCC) of Yunnan University.

Morphological observations

To describe the sexual morphs of the two species, frozen sections or hand sections of the fruiting structures of the stroma were immersed in water and then dyed with lactophenol cotton blue solution for morphological observation and photomicrography (Wang et al. 2021a). For observations on asexual morphs, new colonies were established from old cultures and placed on new PDA plates. The plates were cultured in an incubator for 6 or 12 weeks at 25 °C, and then asexual morphs were observed and recorded (shape, texture, and color of the colonies). Microscope slide cultures were made using the methods of Wang et al. (2020). The morphological observations and measurements were made using Olympus CX40 and BX53 microscopes.

DNA extraction, PCR, and sequencing

Five-centimeter segments from the stroma of fresh specimens and the cultures were used for DNA extraction to ensure the cultures and specimens were the same. Total DNA was extracted using cetyltrimethyl ammonium bromide (CTAB) according to the procedure described by Liu et al. (2001). The DNA was used for PCR amplification. The primer pair, NS4 (5'-CTTCCGTCAATTCCTTTAAG-3') and NS1 (5'-GTAGTCATATGCTTGTCTC-3') was used to amplify nrSSU (the nuclear ribosomal small subunit) (White et al. 1990). The primer pair, LR5 (5'-ATCCTGAGGGAAACTTC-3') and LR0R (5'-GTACCCGCTGAACTTAAGC-3') was used to amplify nr LSU (the nuclear ribosomal large subunit) (Vilgalys and Hester 1990; Rehner and Samuels 1994). The primer pair, 983F (5'-GCYCCYGGHCAYCGTGAYTTYAT-3') and 2218R (5'-ATGACACCRACRGCRACRGTYTG-3') was used to amplify tef-1α (the translation elongation factor 1α) (Rehner and Buckley 2005). The primer pair, CRPB1A (5'-CAYCCWGGYTTYATCAAGAA-3') and RPB1C (5'-CCNGCDATNTCRTTRTCCATRTA-3') were used to amplify rpb1 (the largest subunit of RNA polymerase II) (Castlebury et al. 2004; Bischoff et al. 2006). The primer pair, fRPB2-5F (5'-GAYGAYMGWGATCAYTTYGG-3') and fRPB2-7cR (5'-CCCATRGCTTGYTTRCCCAT-3') was used to amplify rpb2 (the second largest subunit of RNA polymerase II) (Liu et al. 1999). The polymerase chain reaction (PCR) for amplification of the five genes and their sequencing were described by Wang et al. (2015).

Phylogenetic analyses

Sequences of the five genes (nrSSU, nr LSU, tef-1α, rpb1, and rpb2) were downloaded from GenBank, and combined with the newly generated sequences in this study. The taxa information of the species and GenBank accession numbers of the five genes are listed in Table 1. Sequences of the five genes were aligned using the Clustal X (v.2.0) and MEGA6 (v.6.0) (Larkin et al. 2007; Tamura et al. 2013). Ambiguously aligned sites were eliminated, and the gaps were treated as missing data. The aligned sequences of the five genes (nrSSU, nr LSU, tef-1α, rpb1, and rpb2) were concatenated into a single combined dataset using MEGA6 (v.6.0.). Conflicts between the five genes were tested using PAUP* (v.4.0b10) (Swofford 2002). The results of the phylogenetic signals in the five genes were not in conflict. The concatenated dataset containing all five genes consisted of 11 data partitions, including one each for nrSSU and nr LSU, and three for each of the three codon positions of tef-1α, rpb1, and rpb2. Phylogenetic analyses based on the five genes were made using BI and ML methods (Ronquist and Huelsenbeck 2003; Stamatakis et al. 2008). We used the optimal model GTR+I with 1,000 rapid bootstrap replicates on the five genes for ML analyses (Stamatakis 2006). We conducted BI analyses using a GTR+G+I model determined by jModelTest (v.2.1.4), conducted on MrBayes (v.3.1.2) for 5 million generations (Darriba et al. 2012). The phylogenetic tree constructed was viewed and edited using FigTree (v.1.4.2) and Adobe Illustrator CS6.

Table 1.
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Specimen information and GenBank accession numbers of the sequences used in this study.

Species Host Isolate no./ specimen no. GenBank accession no.
nrSSU nr LSU tef-1α rpb1 rpb2
Hirsutella citriformis Cixiidae (Hemiptera) ARSEF 1446 KM652065 KM652106 KM651990 KM652031 –
Hirsutella fusiformis Brachyderes incanus (Curculionidae, Coleoptera) ARSEF 5474 KM652067 KM652110 KM651993 KM652033 –
Hirsutella gigantea Pamphiliidae (Hymenoptera) ARSEF 30 – JX566977 JX566980 KM652034 –
Hirsutella guyana Empoasca kraemeri (Cicadellidae, Hemiptera) ARSEF 878 KM652068 KM652111 KM651994 KM652035 –
Hirsutella illustris Eriosoma lanigerum (Aphididae, Hemiptera) ARSEF 5539 KM652069 KM652112 KM651996 KM652037 –
Hirsutella kirchneri Abacarus hystrix (Eriophyidae, Acari) ARSEF 5551 KM652070 KM652113 KM651997 – –
Hirsutella lecaniicola Parthenolecanium corni (Coccidae, Hemiptera) ARSEF 8888 KM652071 KM652114 KM651998 KM652038 –
Hirsutella liboensis Larva of Cossidae (Lepidoptera) ARSEF 9603 KM652072 KM652115 KY415588 KY945367 –
Hirsutella necatrix Acari ARSEF 5549 KM652073 KM652116 KM651999 KM652039 –
Hirsutella nodulosa Dioryctria zimmermani (Pyralidae, Lepidoptera) ARSEF 5473 KM652074 KM652117 KM652000 KM652040 –
Hirsutella radiata Diptera ARSEF 1369 KM652076 KM652119 KM652002 KM652042 –
Hirsutella rhossiliensis Mesocriconema xenoplax (Criconematidae, Tylenchida) ARSEF 3747 KM652080 KM652123 KM652006 KM652045 –
Hirsutella strigosa Nephotettix virescens (Cicadellidae, Hemiptera) ARSEF 2197 KM652085 KM652129 KM652012 KM652050 –
Hirsutella subulata Microlepidoptae (Lepidoptera) ARSEF 2227 KM652086 KM652130 KM652013 KM652051 –
Hirsutella thompsonii var. synnematosa Aceria sheldoni (Eriophyidae, Acari) ARSEF 2459 KM652099 KM652147 KM652027 KM652061 –
Hirsutella thompsonii var. thompsonii Phyllocoptruta oleivora (Eriophyidae, Acari) ARSEF 137 KM652087 KM652131 KM652014 KM652052 –
Hirsutella thompsonii var. vinacea Acalitus vaccinii (Eriophyidae, Acari) ARSEF 254 KM652101 KM652149 KM652028 KM652062 –
Ophiocordyceps acicularis Larva of Coleoptera OSC 110987 EF468950 EF468805 EF468744 EF468852 –
Ophiocordyceps acicularis Larva of Coleoptera OSC 110988 EF468951 EF468804 EF468745 EF468853 –
Ophiocordyceps agriotidis Larva of Coleoptera ARSEF 5692 DQ522540 DQ518754 DQ522322 DQ522368 DQ522418
Ophiocordyceps annulata Larva of Coleoptera CEM 303 KJ878915 KJ878881 KJ878962 KJ878995 –
Ophiocordyceps aphodii Larva of Scarabaeidae (Coleoptera) ARSEF 5498 DQ522541 DQ518755 DQ522323 – DQ522419
Ophiocordyceps appendiculata Larva of Coleoptera NBRC 106960 JN941728 JN941413 AB968577 JN992462 AB968539
Ophiocordyceps arborescens Larva of Pueraria lobata (Lepidoptera) NBRC 105891 AB968386 AB968414 AB968572 – AB968534
Ophiocordyceps bidoupensis Larva of Elateridae (Coleoptera) YFCC 8793 OM304638 – OK556894 OK556898 OK556900
Ophiocordyceps bidoupensis Larva of Elateridae (Coleoptera) YHH 20036 OK571396 – OK556893 OK556897 OK556899
Ophiocordyceps brunneanigra Cicadellidae (Hemiptera) TBRC 8093 – MF614654 MF614638 MF614668 MF614681
Ophiocordyceps brunneaperitheciata Larva of Lepidoptera TBRC 8100 – MF614658 MF614643 – MF614685
Ophiocordyceps brunneipunctata Larva of Elateridae (Coleoptera) OSC 128576 DQ522542 DQ518756 DQ522324 DQ522369 DQ522420
Ophiocordyceps citrina Hemiptera TNS F18537 – KJ878903 KJ878983 – KJ878954
Ophiocordyceps cochlidiicola Cochlidiidae pupa (Lepidoptera) HMAS 199612 KJ878917 KJ878884 KJ878965 KJ878998 –
Ophiocordyceps cossidarum Larva of Cossidae (Lepidoptera) MFLU 17-0752 MF398186 MF398187 MF928403 MF928404 –
Ophiocordyceps crinalis Larva of Lepidoptera GDGM 17327 KF226253 KF226254 KF226256 KF226255 –
Ophiocordyceps evansii Pachycondyla harpax adult ant (Hymenoptera) HUA 186159 KC610796 KC610770 KC610736 KP212916 –
Ophiocordyceps formicarum Formicidae (Hymenoptera) TNS F18565 KJ878921 KJ878888 KJ878968 KJ879002 KJ878946
Ophiocordyceps forquignonii Adult fly (Diptera) OSC 151902 KJ878912 KJ878876 – KJ878991 KJ878945
Ophiocordyceps furcatosubulata Larva of Elateridae (Coleoptera) YFCC 904 MT774216 MT774223 MT774244 MT774230 MT774237
Ophiocordyceps furcatosubulata Larva of Elateridae (Coleoptera) YHH 17005 MT774217 MT774224 MT774245 MT774231 MT774238
Ophiocordyceps geometridicola Larva of Geometridae (Lepidoptera) TBRC 8095 – MF614648 MF614632 MF614663 MF614679
Ophiocordyceps houaynhangensis Larva of Coleoptera TBRC 8428 – MH092902 MH092894 – –
Ophiocordyceps hydrangea Nymph of cicada (Hemiptera) YFCC 8832 OM304636 OM304640 OM831277 OM831280 OM831283
Ophiocordyceps hydrangea Nymph of cicada (Hemiptera) YFCC 8833 OM304637 OM304641 OM831278 OM831281 OM831284
Ophiocordyceps hydrangea Nymph of cicada (Hemiptera) YFCC 8834 OM304635 OM304639 OM831276 OM831279 OM831282
Ophiocordyceps karstii Hepialus jianchuanensis (Lepidoptera) MFLU:15-3884 KU854952 – KU854945 KU854943 –
Ophiocordyceps kimflemingiae Camponotus castaneus/americanus (Hymenoptera) SC09B KX713631 KX713620 KX713698 KX713724 –
Ophiocordyceps kniphofioides Cephalotes atratus adult ant (Hymenoptera) HUA 186148 KC610790 KF658679 KC610739 KF658667 KC610717
Ophiocordyceps konnoana Larva of Coleoptera EFCC 7315 EF468959 – EF468753 EF468861 EF468916
Ophiocordyceps langbianensis Larva of Coleoptera DL0017 MT928355 MT928306 – – –
Ophiocordyceps lanpingensis Larva of Hepialidae (Lepidoptera) YHOS0705 KC417458 KC417460 KC417462 KC417464 KC456333
Ophiocordyceps longissima Cicada nymph (Cicadidae, Hemiptera) NBRC 106965 AB968392 AB968420 AB968584 – AB968546
Ophiocordyceps longissima Hemiptera; cicada (nymph) EFCC 6814 – EF468817 EF468757 EF468865 –
Ophiocordyceps macroacicularis Larva of Cossidae (Lepidoptera) NBRC 100685 AB968388 AB968416 AB968574 – AB968536
Ophiocordyceps multiperitheciata Lepidoptera larva BCC 69008 – MF614657 MF614641 – MF614682
Ophiocordyceps myrmicarum Hymenoptera (Formicidae) HIRS 45 KJ680150 JX566965 JX566973 KJ680151 –
Ophiocordyceps nigrella Larva of Lepidoptera EFCC 9247 EF468963 EF468818 EF468758 EF468866 EF468920
Ophiocordyceps pruinosa Hemiptera NHJ 12994 EU369106 EU369041 EU369024 EU369063 EU369084
Ophiocordyceps pseudoacicularis Larva of Lepidoptera TBRC 8102 – MF614646 MF614630 MF614661 MF614677
Ophiocordyceps pulvinata Camponotus adult ant (Hymenoptera) TNS-F 30044 GU904208 AB721305 GU904209 GU904210 –
Ophiocordyceps ramosissimum Phassus nodus larva (Lepidoptera) GZUHHN8 KJ028012 – KJ028014 KJ028017 –
Ophiocordyceps ravenelii Beetle larva (Coleoptera) OSC 110995 DQ522550 DQ518764 DQ522334 DQ522379 DQ522430
Ophiocordyceps robertsii Larva of Hepialidae (Lepidoptera) KEW 27083 – EF468826 EF468766 – –
Ophiocordyceps rubiginosiperitheciata Larva of Coleoptera NBRC 106966 JN941704 JN941437 AB968582 JN992438 AB968544
Ophiocordyceps satoi Polyrhachis lamellidens (Hymenoptera) J19 KX713650 KX713601 KX713684 KX713710 –
Ophiocordyceps sinensis Larva of Hepialidae (Lepidoptera) EFCC 7287 EF468971 EF468827 EF468767 EF468874 EF468924
Ophiocordyceps sinensis Larva of Hepialidae (Lepidoptera) YHH 1805 MK984568 MK984580 MK984572 MK984587 MK984576
Ophiocordyceps sobolifera Cicada nymph (Cicadidae, Hemiptera) TNS F18521 KJ878933 KJ878898 KJ878979 KJ879013 –
Ophiocordyceps sobolifera Hemiptera (cicada nymph) NBRC 106967 AB968395 AB968422 AB968590 – –
Ophiocordyceps spataforae Hemiptera adult NHJ 12525 EF469125 EF469078 EF469063 EF469092 EF469111
Ophiocordyceps sphecocephala Hymenoptera adult wasp NBRC 101753 JN941695 JN941446 AB968592 JN992429 AB968553
Ophiocordyceps stylophora Larva of Elateridae (Coleoptera) OSC 110999 EF468982 EF468837 EF468777 EF468882 EF468931
Ophiocordyceps thanathonensis Hymenotera adult ant MFLU 16-2910 MF882926 MF850377 MF872614 MF872616 –
Ophiocordyceps tiputinii Larva of Megaloptera QCNE 186287 KC610792 KC610773 KC610745 KF658671 –
Ophiocordyceps tricentri Adult of Cercopoidea (Hemiptera) NBRC 106968 AB968393 AB968423 AB968593 – AB968554
Ophiocordyceps unilateralis s. str. Camponotus sericeiventris (Hymenoptera) VIC 44303 KX713628 KX713626 KX713675 KX713730 –
Ophiocordyceps unituberculata Larva of Lepidoptera YFCC HU1301 KY923214 KY923212 KY923216 KY923218 KY923220
Ophiocordyceps xuefengensis Larva of Phassus nodus (Lepidoptera) GZUH2012HN14 KC631789 – KC631793 KC631798 –
Ophiocordyceps yakusimensis Cicada nymph (Cicadidae, Hemiptera) HMAS 199604 KJ878938 KJ878902 – KJ879018 KJ878953
Paraisaria amazonica Adult of Acrididae (Orthoptera) HUA 186143 KJ917562 KJ917571 KM411989 KP212902 KM411982
Paraisaria coenomyiae Coenomyia sp. (Coenomyiidae, Diptera) NBRC 106964 AB968385 AB968413 AB968571 – AB968533
Paraisaria gracilis Larva of Lepidoptera EFCC 8572 EF468956 EF468811 EF468751 EF468859 EF468912
Paraisaria heteropoda Cicada nymph (Hemiptera) NBRC 100644 JN941718 JN941423 AB968596 JN992452 AB968557
Tolypocladium inflatum Coleoptera (larva) OSC 71235 EF469124 EF469077 EF469061 EF469090 EF469108
Tolypocladium ophioglossoides Fungi (Elaphomyces sp.) CBS 100239 KJ878910 KJ878874 KJ878958 KJ878990 KJ878944

Results

Phylogenetic analyses

A total of 83 samples were used for the phylogenetic analyses. Five gene sequences of the two new species collected were used to reconstruct the phylogenetic framework of Ophiocordyceps. Two taxa of Tolypocladium were designated as the outgroup, and these were, respectively, Tolypocladium ophioglossoides CBS 100239 and Tolypocladium inflatum OSC 71235. The alignment lengths of the 83 samples were composed of 4,486 bp sequence data, 971 bp of nrSSU, 921 bp of nr LSU, 943 bp of tef-1α, 726 bp of rpb1, and 925 of rpb2. The phylogenetic tree showed that these were identical in overall topologies to previous studies. Four clades (Hirsutella clade, O. sobolifera clade, O. sphecocephala clade, and O. ravenelii clade) of Ophiocordyceps were well-supported by ML bootstrap proportions and BI posterior probabilities (Fig. 1). The two new species in the O. sobolifera clade, O. hydrangea and O. bidoupensis, formed two separate subclades. Three samples of O. hydrangea (BP = 98%, PP = 1) formed a separate subclade with O. longissima and O. yakusimensis, while O. bidoupensis (BP = 83%, PP = 0.99) formed a separate subclade with O. houaynhangensis.

Figure 1. 

Phylogenetic relationships of Ophiocordyceps hydrangea and related species from the five genes dataset (nr LSU, nrSSU, tef-1α, rpb1, and rpb2) based on ML and BI analyses. Statistical support values of BI posterior probabilities and ML bootstrap proportions (0.5/≥50%) are shown at the nodes.

Taxonomy

Ophiocordyceps hydrangea H. Yu, W.Q. Zou & D.X. Tang, sp. nov.

MycoBank No: MycoBank No: 843203
Fig. 2

Etymology

Hydrangea, referred to the top of the stroma similar to hydrangea.

Holotype

China, Yunnan Province, Jinghong City, Nabanhe National Nature Reserve, 22°8'21.32"N, 100°42'18.35"E, alt. 612 m, on cicada nymphs (Cicadidae, Hemiptera). The material was found in the soil of an evergreen broad-leaved forest, 18 August 2020, H. Yu (YHH 20081, holotype; YFCC 8834, ex-holotype culture).

Figure 2. 

Ophiocordyceps hydrangea A, B fungus on a cicada nymph C, D colony on PDA medium E conidiophores, conidiogenous cells and conidia F–J conidiogenous cells and conidia K conidia. Scale bars: 1 cm (A, B); 2 cm (C, D); 10 µm (E, F, G, I, J); 5 µm (H, K).

Sexual morph

The stroma was grown from the head of the host cicada nymph, solitary, the top of the stroma similar to hydrangea, pale pink, 1.6–6.4 cm long. Sexual morph was not observed.

Asexual morph

The colony grew slowly on PDA medium. Cultured at 25 °C for about 12 weeks, the diameter of the colony was 25–28 mm, pale pink, the edge white, hard texture. The back of the colony was white to brown. Surface hyphae rough, hyaline, septate. Conidiophores were cylindrical. Conidiogenous cells were solitary or whorled, ampuliform, smooth-walled, forming on conidiophores or colonies, hyaline, with swollen base, and slender top, 10.6–17.6 µm long, 2.9–4.3 µm wide at the swollen base, and 1.1–2.2 µm wide at the slender top. Conidia hyaline, ovoid or long oval, solitary, 6.8–10.1 × 3.3–4.5 µm.

Host

Cicada nymph (Cicadidae, Hemiptera).

Habitat

In the soil of an evergreen broad-leaved forest.

Distribution

China.

Other material examined

China, Yunnan Province, Jinghong City, Nabanhe National Nature Reserve, 22°8'21.32"N, 100°42'18.35"E, alt. 612 m, on cicada nymphs (Cicadidae, Hemiptera) was found in the soil an evergreen broad-leaved forest, 18 August 2020, H. Yu (YFCC 8832, YFCC 8833).

Notes

Phylogenetic analyses showed that O. hydrangea clustered with O. sobolifera, O. longissima, and O. yakusimensis of the O. sobolifera clade (Fig. 1). Their hosts were cicada nymphs compared to other species of the O. sobolifera clade (Table 2). Ophiocordyceps hydrangea was well supported by BI and ML results, forming a separate subclade with O. sobolifera, O. longissima, and O. yakusimensis. The macro-morphology of O. hydrangea was clearly different from O. sobolifera, O. longissima, O. khonkaenensis, and O. yakusimensis. The stroma of O. hydrangea grew from the head of the host cicada nymph, solitary, and the top of the stroma was like a pale pink hydrangea.

Table 2.
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Morphological comparisons of two new species and related species.

Species Host stromata Perithecia Asci Ascospores Conidiogenous cells Conidia References
O. bidoupensis Larva of Elateridae (Coleoptera) Solitary, solid, cylindrical, yellow, 11.8–22.5 cm long. Immersed, pyriform to lanceolate, brown-yellow, 213.4–405.9 × 74.8–192.4 μm. Hyaline, slender, 116.1–192.7 × 4.8–7.5 μm. Hyaline, filiform, multi-septate. Cone, hyaline, septate, smooth-walled, forming on hyphae, with a hypertrophic base, tapering abruptly into a thin neck, smooth-walled, 13.8–46.4 × 0.42–5.13 μm. Oval or briolette, hyaline, smooth-walled, 2.24–3.61 × 1.49–2.70 μm. This study
O. brunneipunctata Larva of Elateridae (Coleoptera) Solitary, rarely up to 3, simple, 25–90 mm high. Immersed, perithecioid, brown, ovate to pyriform, brown-walled, 270–335 × 110–160 μm. Hyaline, cylindric, capitate, 8-spored, 280–295 × 6–7 μm. Hyaline, filiform, multiseptate breaking into 64 part spores, 4–6 × 1–1.5 μm. Monophialidic, rarely polyphialidic, hyaline, smooth, 5.5–7.5 × 2.5–3.0 μm at the base, up to 15 × 0.5 μm above. Hyaline, aseptate, smooth, spherical 1.5–2.5 μm diam., enveloped by a mucous sheath. Hywel-Jones 1995b; Luangsa-ard et al. 2008
O. cossidarum Larva of Cossidae (Lepidoptera) Solitary, simple, 40–70 mm high. Immersed, red, ovate to phialide, red-walled, 355–454 × 136–171 μm. Hyaline, cylindrical, 8-spored with a thickened apex, 174–221 × 5.7–7 μm. Hyaline, fifiliform, multiseptate,131–153 × 1.8–2.2 μm, breaking into 32 part-spores. – – Hyde et al. 2017
O. furcatosubulata Larva of Elateridae (Coleoptera) Single, solid, yellow to brown, 40–80 mm long, 1.5–2.2 mm wide. Immersed, long ovoid or pyriform, 289.6–405.8 × 87.0–159.2 µm. Hyaline, cylindrical, 138.8–202.5 × 4.3–6.0 μm. Hyaline, filiform, multi-septate, finally breaking into secondary ascospores, 3.7–5.3 × 1.3–2.0 μm. Polyphialidic, forming on conidiophores or side branches, hyaline, with a slender or subulate base, tapering gradually, smooth-walled or verruculose, 3.5–15.8 × 0.9–1.7 μm. Solitary, aseptate, smooth-walled, broadly ellipsoid or ellipsoid, 1.5–2.5 × 1.2–1.9 μm. Wang et al. 2021a
O. houaynhangensis Larva of Coleoptera Solitary, cylindrical, cream, up to 11 cm long and 1.5–2.5 mm in width. Completely immersed, obclavate, 300–450 × 80–170 µm. Cylindrical, 100–250 × 4–7.5 µm. Hyaline, cylindrical, breaking into 32 small truncate part-spores, 4–7 × 1–2 µm. Monophialidic, phialides flasked-shaped with long necks, up to 30 µm long and 2–4 µm in breadth; phialide necks up to 18 μm long and 0.5 µm in breadth. Hyaline, smooth, spherical, 2–3 µm. Crous et al. 2018
O. langbianensis Larva of Coleoptera Solitary, rarely branched, 40–100 mm long. Immersed, ovate or pyriform, 260–400 × 100–190 µm. Cylindrical, with thickened cap, 200–250 × 5.0–6.0 μm. Fliform, multiseptate, articulated in long-chain afer discharging, sometimes breaking into 1-celled part spores, 5–7.5 × 1.3–2 µm. Divergent. Chains, elliptical. Lao et al. 2021
O. sobolifera Cicada nymph (Cicadidae, Hemiptera) Commonly single, rarely fasciculated by twos or threes, arising from head among polster, clavate or cylindric 2–8 cm long, 2–6 mm thick, become hollow after maturity. Rectangularly immersed, ampullaceous 500–600 × 220–260 μm, with somewhat long neck, ostiola somewhat prominent, walls hyaline 8–16 μm thick. Cylindric, 400–470 × 5.6–6.3 μm. Finally breaking into secondary ascospores, truncate at both ends, 6–12 × 1.0–1.3 μm. – Terminal or lateral, ellpsoid or fusiformed, hyaline, 6.5–10.5 × 2.5–4.0 μm. Kobayasi and Shimizu 1963
O. yakusimensis Cicada nymph (Cicadidae, Hemiptera) Very long attaining 14 cm, arising from the apical part between eyes. Wholly embeddèd, narrow ovoid or almost naviculate, 740–800 × 170–230 μm, without protruding ostiola, neck almost destitute, wall 21–23 μm thick, composed of very thin cells. 270–310 × 5 μm. Finally breaking into secondary ascospores, long cylindrical, somewhat attenuated on both sides, terminally truncate, 10–15 × 1 μm. – – Kobayasi and Shimizu 1963
longissima Cicada nymph (Cicadidae, Homoptera) 5–20 cm long, some times much longer. Ovoid to long ovoid, with a short neck, 440–590 × 130–300 µm. 190–350 × 5–6 µm. – – – Sung et al. 2011
O. khonkaenensis Cicada nymph (Hemiptera) Variable in number, solitary to three, 20–30 mm long and 2–3 mm in breath. Immersed, flask shaped, 590–700 × 200–300 µm. Cylindrical, 237.5–337.5 × 5–6 µm. Filiform, 300–360 × 1–1.5 µm readily breaking into 32 part-spores, 7–13 × 1–1.5 µm. Phialidic, hirsutella-like, 5.5–11 × 2–3 µm. Hyaline, fusiform, smoothwalled, 3–5.5 × 1–3 µm. Crous et al. 2019
O. hydrangea Cicada nymph (Cicadidae, Hemiptera) Solitary, the top of the stroma similar to hydrangea, pale pink,1.6–6.4 cm long. – – – Solitary or whorled, ampuliform, smooth-walled, forming on conidiophores or colonies, hyaline, with swollen base, and slender top, 10.6–17.6 µm long, 2.9–4.3 µm wide at the swollen base, and 1.1–2.2 µm wide at the slender top. Hyaline, ovoid or long oval, solitary, 6.8–10.1 × 3.3–4.5 µm. This study

Ophiocordyceps bidoupensis H. Yu, W.Q. Zou & D.X. Tang, sp. nov.

MycoBank No: MycoBank No: 843204
Fig. 3

Etymology

Bidoupensis, referred to the type species collected from Bidoup Nuiba National Park.

Holotype

Vietnam, Lintong Province, Bidoup Nuiba National Park, 12°8'9.30"N, 108°31'51.38"E, alt. 1678 m, on larva of Elateridae (Coleoptera) buried in soil, emerging from the leaf litter on the forest floor, 16 October 2017, H. Yu (YHH 20036, holotype; YFCC 8793, ex-holotype culture).

Figure 3. 

Ophiocordyceps bidoupensis A–C fungus on an Elateridae larva D, E cross-section of the ascoma showing the perithecial arrangement F–H asci I ascospores J, K colony on PDA medium L–N conidiogenous cells and conidia O conidiogenous cells P, Q conidia. Scale bars: 1 cm (A–C); 200 µm (D); 20 µm (E–H); 10 µm (I); 2 cm (J, K); 5 µm (L–Q).

Sexual morph

The stroma grew from the head of the host, solitary, solid, cylindrical, 11.8–22.5 cm long, yellow. Stipe clavate, yellow, curved, 10.7–21.2 cm long, 0.7–0.9 mm wide. Fertile parts cylindrical, yellow, slightly curved, 2.9–11.3 mm long, 0.9–1.6 mm wide. Sterile apices cone, yellow, 2.1–7.2 mm long, 0.2–0.7 mm wide. Perithecia immersed, pyriform to lanceolate, brown-yellow, 213.4–405.9 × 74.8–192.4 μm. Asci hyaline, slender, 116.1–192.7 × 4.8–7.5 μm. Asci cap prominent, capitate, 4.7–6.1 × 3.3–5.4 μm. Ascospores hyaline, filiform, multi-septate.

Asexual morph

The colony grew slowly on PDA medium. Cultured at 25 °C for about 6 weeks, the diameter of the colony was 38–45 mm, white, aerial mycelium on the surface, slightly convex. The back of the colony was grayish-white, dark brown in the middle. Surface smooth of hyphae, hyaline, septate. Conidiogenous cells cone, hyaline, septate, smooth-walled, forming on hyphae, with a hypertrophic base, tapering abruptly to a thin neck, 13.80–46.4 × 0.42–5.13 μm. Conidia hyaline, oval or briolette, smooth-walled, 2.24–3.61 × 1.49–2.70 μm.

Host

Larva of Elateridae (Coleoptera).

Habitat

The hosts were buried in soil, and the stroma were found in the leaf litter on the forest floor.

Distribution

Vietnam.

Notes

Phylogenetic analyses showed that O. bidoupensis was clustered with O. houaynhangensis, O. brunneipunctata, O. langbianensis, O. cossidarum, and O. furcatosubulata of the O. sobolifera clade (Fig. 1). Their hosts were larvae of Elateridae compared to cicada nymph hosts of the other species of the O. sobolifera clade (Table 2). Ophiocordyceos bidoupensis was well-supported by bootstrap support and posterior probabilities, and formed a separate subclade with O. houaynhangensis, O. brunneipunctata, O. langbianensis, and O. cossidarum. The morphology of O. bidoupensis was clearly different in shape and size from other species of O. sobolifera clade (Table 2). The stroma of O. bidoupensis grew solitary from the head of the host; sterile apices of the stroma were different from the other species.

Discussion

Ophiocordyceps is the largest genus in the Ophiocordycipitaceae, with a wide range of hosts and various species. At present, more than 290 species of Ophiocordyceps have been reported (Index Fungorum 2022). However, only 11 species are described in the O. sobolifera clade and their hosts are mainly Coleoptera larvae and cicada nymphs (Hemiptera) (Table 2). We describe the new species O. hydrangea attacking cicada nymphs and the new species O. bidoupensis attacking Coleoptera larvae. Most species have diverse macro-morphological or micro-morphological characteristics due to the same entomopathogenic fungi having a different host, or different species of entomopathogenic fungi having the same host (Sung et al. 2007, 2011; Araújo et al. 2015; Araújo and Hughes 2016; Shrestha et al. 2016; Luangsa-ard et al. 2018; Crous et al. 2019; Fan et al. 2021; Wang et al. 2021a). Hemiptera hosts are widely present among the species of Ophiocordyceps, including species of the Hirsutella clade, O. sobolifera clade, O. sphecocephala clade, and O. ravenelii clade.

The entomopathogenic fungi whose host is Hemiptera have diverse morphological characteristics. For example, O. nutans (Patouillard) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora (Sung et al. 2007), its hosts were stink bugs (Hemiptera), stromata solitary or multiple, fertile parts was red (Hywel-Jones 1995a; Luangsa-ard et al. 2008), stromata of O. brunneinigra (Hemipteran host) were flexuous, arising from between the head and the thorax of the host (Luangsa-ard et al. 2018), stromata of O. spataforae Tasanathai, Thanakipipattana, Khonsanit & Luangsa-ard were cylindrical, cream to pale brown (Luangsa-ard et al. 2018). However, from previously reported Hemipteran hosts, only a few hosts of the O. sobolifera clade were cicada nymphs in Ophiocordyceps (Kobayasi and Shimizu 1963; Sung et al. 2011; Crous et al. 2019). In this study, the host of O. hydrangea was a cicada nymph. More interestingly, the O. hydrangea was significantly more beautiful than other species; the stroma grew from the head of the host cicada nymph, and the top of the stroma like a hydrangea (Sung et al 2007, 2011; Crous et al. 2019). Coleoptera hosts were common in species of Ophiocordyceps. More than 20 species of Ophiocordyceps were parasitic on Coleoptera larvae (Shrestha et al. 2016). These species included O. acicularis (Ravenel) Petch (Petch 1933), O. annulata (Kobayasi & Shimizu) Spatafora, Kepler & C.A. Quandt (Kobayasi and Shimizu 1982; Spatafora et al. 2015), O. aphodii (Mathieson) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora (Mathieson 1949; Sung et al. 2007), O. brunneipunctata (Hywel-Jones) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora (Hywel-Jones 1995b; Sung et al. 2007; Luangsa-ard et al. 2008), O. furcatosubulata H. Yu, Y. Wang & Y.B. Wang (Wang et al. 2021a), O. houaynhangensis Keochanpheng, Thanakitp., Mongkols. & Luangsa-ard (Crous et al. 2018), O. langbianensis T.D. Lao, T.A.H. Le & N.B. Truong (Lao et al. 2021), O. melolonthae (Tulasne & C. Tulasne) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora (Sung et al. 2007), and O. ravenelii (Berkeley & M.A. Curtis) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora (Sung et al. 2007). Most species with Coleopteran host occur in soil and have solid, cylindrical, and yellow stromata. This is consistent with the results of this study.

Phylogenetic analyses based on the data from five genes showed that our phylogenetic framework of Ophiocordyceps was consistent with previous studies (Sung et al. 2007, 2011; Quandt et al. 2014; Simmons et al. 2015; Crous et al. 2018, 2019; Wang et al. 2018, 2021a; Lao et al. 2021). The genus of Ophiocordyceps consists of four clades, including the Hirsutella clade, O. sobolifera clade, O. sphecocephala clade, and O. ravenelii clade. Phylogenetic analyses showed that O. hydrangea clustered with O. sobolifera, O. longissima, and O. yakusimensis in the O. sobolifera clade, and O. bidoupensis clustered with O. houaynhangensis, O. brunneipunctata, O. langbianensis, O. cossidarum, and O. furcatosubulata in the same clade. Species within the O. sobolifera clade had different hosts, and morphological characteristics. These two new species clustered in two separate subclades within the O. sobolifera clade. The hosts of one subclade were cicada nymphs with stromata cylindrical or sarciniform, bright-colored, conidia were macro (Kobayasi and Shimizu 1963; Crous et al. 2019), and the hosts of another subclade were Coleoptera, with stromata cylindrical, conidia small, and a sterile apex on top of the stroma (Hywel-Jones 1995b; Luangsa-ard et al. 2008; Crous et al. 2018; Lao et al. 2021; Wang et al. 2021a). Therefore, the species of the O. sobolifera clade could be divided into two separate subclades when more materials were collected.

The species of O. sobolifera clade had diverse morphological characteristics (Table 2). The entomopathogenic fungi with cicada nymph hosts shared similar characteristics, stromata solitary or multiple, cylindrical, and bright-colored. However, they also differed in morphology. For example, O. sobolifera lacked a protruding ostiole with immersed perithecia (Kobayasi and Shimizu 1963), and this seems to be contrary to O. yakusimensis (Kobayasi and Shimizu 1963). Stromata of O. longissima were longer than other species, and had a short neck in perithecia (Sung et al. 2011). Compared to the ovoid perithecia of O. longissima and O. yakusimensis, O. khonkaenensis was flask-shaped (Crous et al. 2019). The top of the stroma of O. hydrangea was similar to hydrangea, the size and shape of conidiogenous cells and conidia were different from O. khonkaenensis (Table 2). The entomopathogenic fungi using Coleoptera hosts shared similar characteristics, such as stromata solitary, cylindrical, sterile apices on top, bright-colored. However, they had different shape and size of perithecia, asci, ascospores, conidiogenous cells, and conidia. The perithecia of O. bidoupensis was pyriform to lanceolate and brown-yellow. It was similar to O. brunneipunctata, O. furcatosubulata, and O. langbianensis, and only O. houaynhangensis was clavate (Hywel-Jones 1995b; Luangsa-ard et al. 2008; Crous et al. 2018; Lao et al. 2021; Wang et al. 2021a). Conidiogenous cells of O. bidoupensis were cone-shaped, forming on hyphae, with a hypertrophic base, tapering abruptly into a thin neck, smooth-walled, with a smaller thin neck (0.42 µm wide) than O. brunneipunctata (0.5 µm), O. furcatosubulata (0.9 µm), and O. houaynhangensis (0.5 µm).

Due to the unique geographical locations and climate conditions in China and Vietnam, these areas contain a rich species diversity of Cordyceps s.l. However, our survey of Cordyceps s.l. in China and Vietnam only represented a small portion of the total. More samples of Cordyceps s.l. will continue to be collected in China and Southeast Asia in order to uncover additional undescribed taxa, and revise species with the incorrect classification position of this group.

Acknowledgements

This work was funded by the National Natural Science Foundation of China (31870017, 32060007).

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