MycoKeys 92: | 09_| 30 (2022) er-reviewed open-access journal doi: 10.3897/mycokeys.92.86 | 60 < Mycokeys https://mycokeys.pensoft. net Launched to accelerate biodiversity research Multigene phylogeny and morphology reveal Ophiocordyceps hydrangea sp. nov. and Ophiocordyceps bidoupensis sp. nov. (Ophiocordycipitaceae) Weiqiu Zou! , Dexiang Tang!?,, Zhihong Xu', Ou Huang", Yuanbing Wang', Ngoc-Lan Tran’, Hong Yu! | Yunnan Herbal Laboratory, College of Ecology and Environmental Sciences, Yunnan University, Kunming 650504, Yunnan, China 2 School of Life Science, Yunnan University Kunming 650504, Yunnan, China 3 Institute of Regional Research and Development, Ministry of Science and Technology, Hanoi, Vietnam Corresponding author: Hong Yu (hongyu@ynu.edu.cn, herbfish@163.com) Academic editor: Cecile Gueidan | Received 5 May 2022 | Accepted 10 August 2022 | Published 30 August 2022 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 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 re- constructed using a multigene (nrSSU, nrLSU, tef-1a, 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 * Those authors contributed equally to this work. Copyright Weigiu Zou et al. 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. 110 Weigiu Zou et al. / MycoKeys 92: 109-130 (2022) 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 es- tablished 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 sec- ondary 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 Clavicipi- taceae. 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 re- search results of Sung et al. (2007) are currently the most widely accepted phylogenetic classification of Cordyceps s.1. 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 bee- tle larvae to social insects. More than 10 insect orders were attacked, including Hemiptera, Coleoptera, Lepidoptera, Blattaria, Dermaptera, Diptera, Hymenop- tera, Isoptera, Megaloptera, and Mantodea (Araujo et al. 2015; Araujo and Hughes 2016, 2019). Entomopathogenic fungi whose hosts are cicada nymphs have at- tractive stromata. The most typical representative of this group was Cordyceps ci- cadae (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 & Luang- sa-ard (Crous et al. 2019), O. sobolifera (Hill ex Watson) G.H. Sung, J.M. Sung, Multigene phylogeny and morphology of two new species 111 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 re- ported 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. langshanensis (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. pingbi- anensis 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.1. 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 un- known species of Ophiocordyeps attacking a cicada nymph was collected from Yun- nan Province, Jinghong City, Nabanhe National Nature Reserve, in China. Another 112 Weigiu Zou et al. / MycoKeys 92: 109-130 (2022) Table |. Specimen information and GenBank accession numbers of the sequences used in this study. Species Hirsutella citriformis Hirsutella fusiformis Hirsutella gigantea Hirsutella guyana Hirsutella illustris Hirsutella kirchneri Hirsutella lecaniicola Hirsutella liboensis Hirsutella necatrix Hirsutella nodulosa Hirsutella radiata Hirsutella rhossiliensis Hirsutella strigosa Hirsutella subulata Hirsutella thompsonii vat. synnematosa Hirsutella thompsonii vat. thompsonii Hirsutella thompsonii vat. vinacea Ophiocordyceps acicularis Ophiocordyceps acicularis Ophiocordyceps agriotidis Ophiocordyceps annulata Ophiocordyceps aphodi Ophiocordyceps appendiculata Ophiocordyceps arborescens Ophiocordyceps bidoupensis Ophiocordyceps bidoupensis Ophiocordyceps brunneanigra Ophiocordyceps brunneaperitheciata Ophiocordyceps brunneipunctata Ophiocordyceps citrina Ophiocordyceps cochlidiicola Ophiocordyceps cossidarum Ophiocordyceps crinalis Ophiocordyceps evansii Ophiocordyceps formicarum Host Cixiidae (Hemiptera) Brachyderes incanus (Curculionidae, Coleoptera) Pamphiliidae (Hymenoptera) Empoasca kraemeri (Cicadellidae, Hemiptera) Eriosoma lanigerum (Aphididae, Hemiptera) Abacarus hystrix (Eriophyidae, Acari) Parthenolecanium corni (Coccidae, Hemiptera) Larva of Cossidae (Lepidoptera) Acari Dioryctria zimmermani (Pyralidae, Lepidoptera) Diptera Mesocriconema xenoplax (Criconematidae, Tylenchida) Nephotettix virescens (Cicadellidae, Hemiptera) Microlepidoptae (Lepidoptera) Aceria sheldoni (Eriophyidae, Acari) Phyllocoptruta oleivora (Eriophyidae, Acari) Acalitus vaccinii (Eriophyidae, Acari) Larva of Coleoptera Larva of Coleoptera Larva of Coleoptera Larva of Coleoptera Larva of Scarabaeidae (Coleoptera) Larva of Coleoptera Larva of Pueraria lobata (Lepidoptera) Larva of Elateridae (Coleoptera) Larva of Elateridae (Coleoptera) Cicadellidae (Hemiptera) Larva of Lepidoptera Larva of Elateridae (Coleoptera) Hemiptera Cochlididae pupa (Lepidoptera) Larva of Cossidae (Lepidoptera) Larva of Lepidoptera Pachycondyla harpax adult ant (Hymenoptera) Formicidae (Hymenoptera) Isolate no./ specimen no. ARSEF 1446 ARSEF 5474 ARSEF 30 ARSEF 878 ARSEF 5539 ARSEF 5551 ARSEF 8888 ARSEF 9603 ARSEF 5549 ARSEF 5473 ARSEF 1369 ARSEF 3747 ARSEF 2197 ARSEF 2227 ARSEF 2459 ARSEF 137 ARSEF 254 OSC 110987 OSC 110988 ARSEF 5692 CEM 303 ARSEF 5498 NBRC 106960 NBRC 105891 YFCC 8793 YHH 20036 TBRC 8093 TBRC 8100 OSC 128576 TNS F18537 HMAS 199612 MEPLU 17-0752 GDGM 17327 HUA 186159 TNS F18565 nrSSU KM652065 KM652067 KMG652068 KM652069 KMG652070 KM652071 KM652072 KM652073 KM652074 KM652076 KM652080 KM652085 KM652086 KM652099 KM652087 KM652101 EF468950 EF468951 DQ522540 KJ878915 DQ522541 JN941728 AB968386 OM304638 OK571396 DQ522542 KJ878917 MF398186 KF226253 KC610796 KJ878921 GenBank accession no. orLSU KM652106 KM652110 JX566977 KM652111 KM652112 KM652113 KM652114 KM652115 KM652116 KM652117 KM652119 KM652123 KM652129 KM652130 KM652147 KM652131 KM652149 EF468805 EF468804 DQ518754 KJ878881 DQ518755 JN941413 AB968414 MF614654 MF614658 DQ518756 KJ878903 KJ878884 MF398187 KF226254 KC610770 KJ878888 tef-1u KM651990 KM651993 JX566980 KM651994 KM651996 KM651997 KM651998 KY415588 KM651999 KM652000 KM652002 KM652006 KM652012 KM652013 KM652027 KM652014 KM652028 EF468744 EF468745 DQ522322 KJ878962 DQ522323 AB968577 AB968572 OK556894 OK556893 MF614638 MF614643 DQ522324 KJ878983 KJ878965 MF928403 KF226256 KC610736 KJ878968 rpbl rpb2 KM652031 - KM652033 = KM652034 - KM652035 = KM652037 = KMG652038 - KY945367 = KM652039 - KM652040 - KM652042 - KM652045 - KM652050 - KM652051 - KM652061 - KM652052 - KM652062 - EF468852 = EF468853 = DQ522368 DQ522418 KJ878995 - L DQ522419 JN992462 AB968539 - AB968534 OK556898 OK556900 OK556897 OK556899 MFG614668 MF614681 = MEG14685 DQ522369 DQ522420 e KJ878954 KJ878998 © MF928404 - KF226255 a KP212916 = KJ879002 KJ878946 Multigene phylogeny and morphology of two new species Species Host Ophiocordyceps forquignonii Adult fly (Diptera) Ophiocordyceps Larva of Elateridae Jurcatosubulata (Coleoptera) Ophiocordyceps Larva of Elateridae Jurcatosubulata (Coleoptera) Ophiocordyceps geometridicola Larva of Geometridae (Lepidoptera) Ophiocordyceps Larva of Coleoptera houaynhangensis Ophiocordyceps hydrangea Nymph of cicada (Hemiptera) Ophiocordyceps hydrangea Nymph of cicada (Hemiptera) Ophiocordyceps hydrangea Nymph of cicada (Hemiptera) Ophiocordyceps Rarstii Hepialus jianchuanensis (Lepidoptera) Ophiocordyceps kimflemingiae Camponotus castaneus/ americanus (Hymenoptera) Ophiocordyceps kniphofioides — Cephalotes atratus adult ant (Hymenoptera) Ophiocordyceps konnoana Larva of Coleoptera Ophiocordyceps langbianensis Larva of Coleoptera Ophiocordyceps lanpingensis Larva of Hepialidae (Lepidoptera) Ophiocordyceps longissima Cicada nymph (Cicadidae, Hemiptera) Ophiocordyceps longissima Hemiptera; cicada (nymph) Ophiocordyceps Larva of Cossidae macroacicularis (Lepidoptera) Ophiocordyceps Lepidoptera larva multiperitheciata Ophiocordyceps myrmicarum Hymenoptera (Formicidae) Ophiocordyceps nigrella Larva of Lepidoptera Ophiocordyceps pruinosa Hemiptera Ophiocordyceps Larva of Lepidoptera pseudoacicularis Ophiocordyceps pulvinata Camponotus adult ant (Hymenoptera) Ophiocordyceps ramosissimum Phassus nodus larva (Lepidoptera) Ophiocordyceps ravenelii Beetle larva (Coleoptera) Ophiocordyceps robertsii Larva of Hepialidae (Lepidoptera) Ophiocordyceps Larva of Coleoptera rubiginosiperitheciata Ophiocordyceps satoi Polyrhachis lamellidens (Hymenoptera) Ophiocordyceps sinensis Larva of Hepialidae (Lepidoptera) Ophiocordyceps sinensis Larva of Hepialidae (Lepidoptera) Ophiocordyceps sobolifera Cicada nymph (Cicadidae, Hemiptera) Ophiocordyceps sobolifera Hemiptera (cicada nymph) Ophiocordyceps spataforae Hemiptera adult Ophiocordyceps sphecocephala Hymenoptera adult wasp Larva of Elateridae (Coleoptera) Hymenotera adult ant Ophiocordyceps stylophora Ophiocordyceps thanathonensis Isolate no./ specimen no. OSC 151902 YFCC 904 YHH 17005 TBRC 8095 TBRC 8428 YFCC 8832 YECC 8833 YECC 8834 MFLU:15-3884 SCO9B HUA 186148 EFCC 7315 DL0017 YHOS0705 NBRC 106965 EFCC 6814 NBRC 100685 BCC 69008 HIRS 45 EFCC 9247 NHJ 12994 TBRC 8102 TNS-F 30044 GZUHHN8 OSC 110995 KEW 27083 NBRC 106966 y9 EFCC 7287 YHH 1805 TNS F18521 NBRC 106967 NHJ 12525 NBRC 101753 OSC 110999 MELU 16-2910 nrSSU KJ878912 MT1774216 MT1774217 OM304636 OM304637 OM304635 KU854952 KX713631 KC610790 EF468959 MT928355 KC417458 AB968392 AB968388 KJ680150 EF468963 EU369106 GU904208 kJ028012 DQ522550 JN941704 KX713650 EF468971 MK984568 KJ878933 AB968395 EF469125 JN941695 EF468982 MF882926 GenBank accession no. orLSU KJ878876 MT774223 M1774224 MF614648 MH092902 OM304640 OM304641 OM304639 KX713620 KF658679 MT928306 KC417460 AB968420 EF468817 AB968416 MF614657 JX566965 EF468818 EU369041 MF614646 AB721305 DQ518764 EF468826 JN941437 KX713601 EF468827 MK984580 KJ878898 AB968422 EF469078 JN941446 EF468837 MF850377 tef-1u. MT774244 MT774245 MF614632 MH092894 OM831277 OM831278 OM831276 KU854945 KX713698 KC610739 EF468753 KC417462 AB968584 EF468757 AB968574 MF614641 JX566973 EF468758 EU369024 MF614630 GU904209 kJo28014 DQ522334 EF468766 AB968582 KX713684 EF468767 MK984572 KJ878979 AB968590 EF469063 AB968592 EF468777 MF872614 rpbl kJ878991 M1774230 M1774231 MF614663 OM831280 OM831281 OM831279 KU854943 KX713724 KF658667 EF468861 KC417464 EF468865 KJ680151 EF468866 EU369063 MF614661 GU904210 KJ028017 DQ522379 JN992438 KX713710 EF468874 MK984587 KJ879013 EF469092 JN992429 EF468882 MF872616 Ths rpb2 KJ878945 M1774237 MT1774238 MF614679 OM831283 OM831284 OM831282 KC610717 EF468916 KC456333 AB968546 AB968536 MF614682 EF468920 EU369084 MF614677 DQ522430 AB968544 EF468924 MK984576 EF469111 AB968553 EF468931 114 Weiqiu Zou et al. / MycoKeys 92: 109-130 (2022) Species Host Isolate no./ GenBank accession no. specimen no. nrSSU nt LSU tef-1e. rpb1 rpb2 Ophiocordyceps tiputinii Larva of Megaloptera QCNE 186287 KC610792 KC610773 KC610745 KF658671 - Ophiocordyceps tricentri Adult of Cercopoidea NBRC 106968 —_AB968393 AB968423 AB968593 - AB968554 (Hemiptera) Ophiocordyceps unilateralis Camponotus sericeiventris VIC 44303 KX713628 KX713626 KX713675 KX713730 - s. st. (Hymenoptera) Ophiocordyceps unituberculata Larva of Lepidoptera YFCC HU1301 KY923214 KY923212 KY923216 KY923218 KY923220 Ophiocordyceps xuefengensis Larva of Phassus nodus GZUH2012HN14 KC631789 - KC631793 KC631798 - (Lepidoptera) Ophiocordyceps yakusimensis Cicada nymph (Cicadidae, HMAS 199604 — kjJ878938 —kJ878902 - kJ879018 KJ878953 Hemiptera) Paraisaria amazonica Adult of Acrididae HUA 186143 KJ917562.—-KJ917571 =KM411989 KP212902 KM411982 (Orthoptera) Paraisaria coenomyiae Coenomyia sp. NBRC 106964 AB968385 AB9G68413 AB968571 - AB968533 (Coenomyiidae, Diptera) Paraisaria gracilis Larva of Lepidoptera EFCC 8572 EF468956 EF468811 EF468751 EF468859 EF468912 Paraisaria heteropoda Cicada nymph NBRC 100644 JN941718 JN941423 AB968596 JN992452 AB968557 (Hemiptera) Tolypocladium inflatum Coleoptera (larva) OSC 71235 EF469124 EF469077 EF469061 EF469090 EF469108 Tolypocladium ophioglossoides Fungi (Elaphomyces sp.) CBS 100239 KJ878910 KJ878874 KJ878958 KJ878990 _‘KJ878944 unknown species of Ophiocordyeps attacking larvae of Elateridae was collected from Lintong Province, Bidoup Nuiba National Park, in Vietnam. The phylogeny and mor- phology of these two fungi were determined, and their systematic position was estab- lished 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. Multigene phylogeny and morphology of two new species LS Morphological observations To describe the sexual morphs of the two species, frozen sections or hand sec- tions 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 ampli- fication. The primer pair, NS4 (5'-CTTCCGTCAATTCCTTTAAG-3') and NS1 (5'-GTAGTCATATGCTTGTCTC-3') was used to amplify nrSSU (the nuclear ribo- somal small subunit) (White et al. 1990). The primer pair, LR5 (5'-ATCCTGAGG- GAAACTTC-3') and LROR (5'-GTACCCGCTGAACTTAAGC-3') was used to amplify nrLSU (the nuclear ribosomal large subunit) (Vilgalys and Hester 1990; Reh- ner and Samuels 1994). The primer pair, 983F (5'-GCYCCYGGHCAYCGTGAY- TTYAT-3') and 2218R (5'-ATGACACCRACRGCRACRGTYTG-3') was used to amplify zef-J« (the translation elongation factor 1x) (Rehner and Buckley 2005). The primer pair, CRPB1A (5'-CAYCCWGGYTTYATCAAGAA-3') and RPBIC (5'-CC- NGCDATNTCRITRICCATRIA-3') were used to amplify 7p4/ (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'-CCC- ATRGCTTGYTTRCCCAT-3') was used to amplify 7p62 (the second largest subunit of RNA polymerase II) (Liu et al. 1999). The polymerase chain reaction (PCR) for am- plification of the five genes and their sequencing were described by Wang et al. (2015). Phylogenetic analyses Sequences of the five genes (nrSSU, nrLSU, tef-1a, 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 Table1. Sequences of the five genes were aligned using the Clustal X (v.2.0) and MEGAG (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, nrLSU, tef-1a, rpb1, and rpb2) were concatenated into a single 116 Weigiu Zou et al. / MycoKeys 92: 109-130 (2022) combined dataset using MEGAG (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 con- sisted of 11 data partitions, including one each for nrSSU and nrLSU, and three for each of the three codon positions of tef-1a, 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 boot- strap replicates on the five genes for ML analyses (Stamatakis 2006). We conducted BI analyses using a GIR+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 Fig Iree (v.1.4.2) and Adobe Illustrator CS6. 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 nrLSU, 943 bp of tef-1a, 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 sepa- rate 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. Taxonomy Ophiocordyceps hydrangea H. Yu, W.Q. Zou & D.X. Tang, sp. nov. MycoBank No: 843203 Fig. 2 Etymology. Hydrangea, referred to the top of the stroma similar to hydrangea. Holotype. Curna, 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). Multigene phylogeny and morphology of two new species ie i100 Ophiocordycep 0.81/8 Ophiocordyceps si 0.93/57 Ophiocordyceps 0.99/22Mleme Ophiocordyceps robe 0.63/83 Ophiocordyceps : et | Ophiocordyceps lanp! BINA Ophiocordyceps mi Hirsutella page i100 OPhiocor 1/68| Hirsutella. SHE q Ophiocordyceps 0.97/62 iT Ophiocordyceps app Ophiocordyceps si oof Ophiocordyce Ophiocordyceps aci i100 Ophiocordyceps arborescens Ophiocordyceps ramosis. Hirsutella rhossiliensis A Hirsutella lecaniicolc Ophiocordyceps. Hirsutella kirchneri ARSEF 5551 Ophiocordyceps crinalis GDGM 17327 Ophiocordyceps brunneanigra TBRC 8093 Ophiocordyceps spataforae NHJ 12525 Hirsutella guyana ARSEF 878 phic east pruinosa Buu 12994 0.78/35 saat 0.65/30 7 0.99/43 0.61/59 1/100} 0.99/99] Ophiocordyceps pseudoa 0ONL Hirsutella nodulosa ARSEF 5473 onan Hirsutella subulata ARSEF 2227 Ophiocordyceps brunneaperitheciata TBRC 8100 Ophiocordyceps agriotidis ARSEF 5692 Hirsutella necatrix ARSEF 5549 ON oo 10of/Zirsutella thompsonii var. vinacea ARSEF 254 1/1008 Hirsutella thompsonii var. thompsonii ARSEF 137 Hirsutella thompsonii var. synnematosa ARSEF 2459 a Ophiocordyceps pulvinata TNS-F 30044 Ophiocordyceps satoi J19 0.99/84 aa. . ‘ial 1/100 Ophiocordyceps unilateralis VIC 44303 Hirsutella ant Ophiocordyceps kimflemingiae SC09B pathogen subclade Ophiocordyceps tiputinii QCNE 186287 Ophiocordyceps kniphofioides HUA 186148 Hirsutella citriformis ARSEF 1446 at ris Hirsutella gigantea ARSEF 30 woop Hirsutella radiata ARSEF 1369 Hirsutella fusiformis ARSEF 5474 Hirsutella liboensis ARSEF 9603 = Ophiocordyceps myrmicarum HIRS 45 af OPHiocordyceps ; 0.99/87 = Ophiocordyceps h 1/98 Ophiocordyceps hyd Be |_| Ophiocordyceps longissima a 100 al Ophiocordyceps longiss: aa | | Ophiocordyceps yakus L_ Ophiocordyceps sobolifer Ophiocordyceps sobolife 1/100} Ophiocordyceps 0.99/83] H. thompsonii subclade 0.57/12 1/93 0.60/53 0.96/44. 1/100} — Ophiocordyceps konnoana EFCC 7 1/1001 Ophiocordyceps ravenelii OSC 1 Mb] _ Lophiocordveps nigra BCC 924 Paraisaria gracilis EFCC 8572 Paraisaria amazonica HUA 186143 | Paraisaria PT ee NBRC 100644 | 100 Tol) pease inflatum OSC Toa Tolypocladium ophioglossoides CBS 100239 0.02 —_ Figure |. Phylogenetic relationships of Ophiocordyceps hydrangea and related species from the five genes dataset (nrLSU, nrSSU, tef-1a, rpb1, and rpb2) based on ML and BI analyses. Statistical support values of BI posterior probabilities and ML bootstrap proportions (0.5/250%) are shown at the nodes. 118 Weigiu Zou et al. / MycoKeys 92: 109-130 (2022) 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, 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 um (E, FG, I, J); 5 pm (H, K). Multigene phylogeny and morphology of two new species 119 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 um long, 2.9—4.3 um wide at the swol- len base, and 1.1—2.2 um wide at the slender top. Conidia hyaline, ovoid or long oval, solitary, 6.8-10.1 x 3.3-4.5 um. Host. Cicada nymph (Cicadidae, Hemiptera). Habitat. In the soil of an evergreen broad-leaved forest. Distribution. China. Other material examined. Cuina, 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 for- est, 18 August 2020, H. Yu (YFCC 8832, YFCC 8833). Notes. Phylogenetic analyses showed that O. hydrangea clustered with O. sobolif- era, 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. khonkaenen- sis, 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. Ophiocordyceps bidoupensis H. Yu, W.Q. Zou & D.X. Tang; sp. nov. 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, emerg- ing from the leaf litter on the forest floor, 16 October 2017, H. Yu (YHH 20036, holo- type; YFCC 8793, ex-holotype culture). Sexual morph. The stroma grew from the head of the host, solitary, solid, cylin- drical, 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 x 74.8- 192.4 um. Asci hyaline, slender, 116.1-192.7 x 4.8-7.5 um. Asci cap prominent, capitate, 4.7—6.1 x 3.3-5.4 um. 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 myce- lium 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, 120 Weigiu Zou et al. / MycoKeys 92: 109-130 (2022) tapering abruptly to a thin neck, 13.80—46.4 x 0.42—5.13 um. Conidia hyaline, oval or briolette, smooth-walled, 2.24—3.61 x 1.49-2.70 um. 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. Figure 3. Ophiocordyceps bidoupensis A—C fungus on an Elateridae larva D, E cross-section of the ascoma showing the perithecial arrangement FH asci I ascospores J, K colony on PDA medium L=N conidiog- enous cells and conidia O conidiogenous cells P, Q conidia. Scale bars: 1 cm (A=C); 200 um (D); 20 um (E-H); 10 um (1); 2 cm J, K); 5 um (L-Q). Multigene phylogeny and morphology of two new species 124 Notes. Phylogenetic analyses showed that O. bidoupensis was clustered with O. houaynhangensis, O. brunneipunctata, O. langbianensis, O. cossidarum, and O. furca- tosubulata of the O. sobolifera clade (Fig. 1). Their hosts were larvae of Elateridae com- pared 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. brunnei- punctata, 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 spe- cies 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; Aratijo et al. 2015; Araujo 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 beauti- ful 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 109-130 (2022) Weigiu Zou et al. / MycoKeys 92 122 £961 nziuityg pue Isekeqoyy €961 nziuiyg pue Isekeqoyy IZOC Je 19 OVT 8107 ‘Je 19 sno) PT COC ‘ye 19 Suey L107 Te 19 opAEy 8007 Te 9 pre-esSuen’] “45661 souof-JamApy Apmis srqy, SIOUIIIFOY vurtl 0°7-G°7 x SOI-<'9 ouTpedy “pourojisny 10 plosdyja qeraqe] JO [eUTUNIOT, ‘Teondiyja ‘suey ‘ul ¢-7 ‘qeorsayds ‘moous ‘ouTpeApy ‘um 6°1-Z'T x ¢°7—C'T “prosdiyje ro prosdiyja ATpeorq “poypem-toours ‘oreadase ‘AreN]OS “yaeays snoonur v £q podoypaaua “urerp wl ¢°7—-G"] yeouayds ‘tnoours ‘oneadase ‘outpeApy “wal Q/°7-GF'T * I9EVTT “partes -yoours ‘aureAy ‘aMIOLIg IO TRAC) vIpruo+) Lo “yapearg ur wm ¢9 pure suoj um g] 09 dn sysou apryeryd stppeaiq ur um 7-7 pure Suc] ui 9¢ 03 dn ‘sypou Bu0] tpim podeys-paysep sapreryd ‘orprerydouoyy url /"1-6'0 X B°GI-G'€ “osopNONIAA JO payem-yoours ‘Ajjenpes3 Sursadei ‘oseq axejnqns Jo Jopugys & YIM ‘ouTpeAY ‘soyueI apts JO soroydorpruos uo Suru0y Orprperyddjog ‘aaoge wi ¢°Q x GT 01 dn ‘aseq ay) ye wl Q'¢-G'z x ¢'/-G'¢ “Moours suredy SOrpreryddjod Ajarex ‘orprperydouoyy “wn €T'C-ZF'0 X F'9OF-8'E I “Pryfes-oours Spoeu ulp e oyur Apdnige Surredey “seq srydonsoddy v& yr ovyddy uo Sururz0y ‘pae4-tpoours ‘ye3das ‘Sourpedy “ou07) s]]J29 snousSorpruo’) “ul [ x CI-O1 ‘ayeounn ATeurura} ‘sapts qiog UO parenuone yeyMourOs qeorrpuryéo Buoy ‘sorodsoose Arepuooes o1ur Supyearq ATpeury "um €*[-0'T x ZI-9 ‘spus tog we sesuNI} ‘sarodsoose Arepuooes o1ur Supyeorq ATpeury ‘unl 7-€"T x ¢°/—¢ ‘sasods aed payjeo-] ort Supyearq soummouros ‘Bursreypsip Joye ureyo-Suo] Ur porepnone ‘oreidasnynur “WIIOFTPT ‘wml 7] x /-4 ‘sorods -ured ayeounn [jews TE O1UT Supyearq ‘jeorpurAo ‘ourpeApy “umn 0'7-€'] x €'C-L’¢ ‘sarodsoose Arepuodes ovur Supyearg Aypeuy ‘oxe1das “Bynuy “unos Ty ourpeAEY ‘sorods-red ZE OyuT Suryearq ‘ud 7°7—-g"T x CCI-[E] o1eidasnnur ‘uOF YY ‘ourpeapy uml CT-T x O-F ‘sorods aed 9 o1ur Bunyearq areadasnynur ‘unroxy ‘ourpeAEy ‘ayeadas “pnw w0pTy oureAy] sarodsoosy "ul ¢ x OLE-OLZ “url €9-9'¢ x OLF-00F OuputD ‘um 09 —0°S * 0S7—007 ‘deo pouaxpryp TM qeoupurya) "ual ¢*/-4 x 0ST —O0T eopuysy ul 09-€'F x C'COC-8'8EI qeotrpuryéo ‘ourpeApy win /- 2° x ICC-PLI ‘xode pousypryy B YM parods-g qeotrpuryéo ‘ourpeApEy ruatl /-9 x 667-087 ‘parods-g ‘oreudeo ‘ourpurpd ‘ourpeAEy “unl C*Z-8°F x LTOI-V9OIT Japueys ourpeAyy *“syjeo ury ATA jo posoduros ‘yoru wal ¢7—-1Z Je inansep asourye you “efonso Surpnnord oy “uM YE7-OZT xX 0O8-OPZL ‘IeTNoTAvu Asowye IO ploao Morreu peppoquis AOU ‘yor wl g[-g ouTedy syfem AuauTurord JYMOUIOS FTONSO “you Buoy yeyMouros ym “tur Y97-Y7TZ x 009-006 snosoryjndure ‘posrourunt Ayrepnsuew2y “wrt OGI-OOT * 0OF-09Z ‘uarosiiAd 10 ayeAo “passouTUy “url QZ 1-08 x 0SF-00E ‘areARpqo “posIouruT Approduro7) “wl 7°6S1 0°28 X 8°S0F—-9'687 “wHOFTAd JO ploAo Buoy ‘pasiouTU] ‘wi T/1-9€T x POH-CCE “payjem-poas ‘apreryd O} 9J€AO “par “posIouruuy "wil OOT-OLT x SEE-OL7 ‘poypem-umorg ‘turox114d 01 o1eA0 ‘uMoIg ‘properpiied ‘pasrouruy “url $76 1-8°FL * 6'SOF-F'E1Z ‘moy]e4-UMOIg ‘ayeTOSI UE] 0} waoys4d ‘pasrowwy] epoyWag ‘soko uaamioq wed yeorde | (eroadruopy Seprpesiy) ydurdu epesry op wo Bursire “Wd FT Sururene Suoy A190, ‘ALMICUT JIE MOT[OY sur090q “ySTYy) WU 9-7 ‘Buoy Wd g—Z STIpuT}Ad JO aIearp Joisjod 3uowe peoy wos Bursie ‘soar | (eroidruropy ‘SEPIPEOT) Ajerer ‘o]3urs ApuourIO7) | Ydurcu eprsiy Io som) Aq parernonsey viaidozjo7 jo vAIeT “‘Suo] wu 00 T-0F ‘poypurrq Ajorer ‘Arenyjos “UPI Ul uur ¢°7—-G"] pure Suoy wid [[ 01 dn “ureayo viaidosjo7 qeotrpurydo ‘Areiyjos jo PATE] (eraidosjo7) seplioreyy jo PAIeT “SPTAA UU, T°7—G"T ‘Buoy WU (g—-h ‘UMOIq 0} MOTs ‘pros opsuIS (exoadoprdaq) “ysty wu Q/ aePIssoc) —0F ‘oydums Areryos jo PAIeT] (era1doajo7) sepHoreyy jo vAIeT "ysry wr Q6—GZ ‘oydumts “¢ o1 dn Ajares ‘Arenyjos (vsa3do2]07)) sepHoreyy jo PAIET *‘Suo] Wo S77-8' IT ‘moqped TeotpurpAd “pros ‘Azex1jo PyeWOIS sasuaueisnevll ‘CE mafyogos ‘CQ SISUIUDIQBUY] ‘C) sisuasupqudpnog O vynnqnsoqwoinf Oo UNAD PISS 0) ‘CEC vypjound1auun.g a) sisuadnop7q ‘O so1edg ‘satads parejas pue saqsads mau 0M Jo suostieduroo [es1sojoydsopy *7 BIQUL 123 Multigene phylogeny and morphology of two new species *do} Jopuays ayy ye opras wd 7°7-]°T pue ‘aseq UaTJOMs oY} 7B api uw €F-6'C ‘wil CF-€'E x ‘Buoy wm 9°/ [-9'0] ‘doy sopusays pue ‘Suo] wo F°9-9' [ud | (erardruazy T'01-8'9 ‘Arenyjos ‘aseq UaTTOMAS YIM ‘ouTeAY ‘saTuOTOS ayed ‘eaSuespdy 0} ‘oepIpeoiy) qeao 3uo] 10 Jo saroydorpruos uo Surunsoy ‘payyem. Je]IUNIs LUIONS 34) ydurku Apmis sryy, | proso ouredzy =| -qioours ‘w0xNdure ‘payroy so AreyTOS jo do} ay) ‘Asenyos epeory vasuviply ‘OQ "yyearg UT TU ¢-Z ‘umn ¢-] x ¢G-€ ‘un ¢*[-] x €[—Z ‘sorods pure suo] urur 9¢—-9Z 6107 ‘poyfemuoours -red 7¢ o1ur Buryeaigq Appear ‘uml 9-¢ x C*ZE¢ ‘un QO¢E-00T x ‘sary 01 Areqyjos (esoidtu3 Fy) sisuauanyuogy ‘Je I9 snoD | ‘wAOFIsny ‘auTpeAEY | ‘wl C-Z x TI- | 0OZ-06S ‘podeys ysepy ‘pasrourUT] goquinu ur gqeueA — | ydurdu epeoiy O (eroidow0py L1OZ ‘un QOE-OET x 06S-OFF Bou ‘ToSU0] YONuT sow “SePIPeoid) ‘ye 10 3ung - ‘unl Q—G x QSE-OG6I | WOYS & YM ‘ploao Buoy 01 ploag | sutos ‘Buoy wo gz7—-<¢ | ydurdu epesry DULISSIBUO] CQ) SUsIOFOY eIpruoy s][29 snouasorpru0; | ss sagodsoosy BDIYWIAg PyEWONS so1adg 124 Weiqiu Zou et al. / MycoKeys 92: 109-130 (2022) (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. furcatosub- ulata H. Yu, Y. Wang & Y.B. Wang (Wang et al. 2021a), O. houaynhangensis Keochan- pheng, Thanakitp., Mongkols. & Luangsa-ard (Crous et al. 2018), O. langbianensis'T.D. Lao, TLA.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 phylo- genetic 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. rav- enelii clade. Phylogenetic analyses showed that O. hydrangea clustered with O. sobolif- era, 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 dif- ferent 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. How- ever, 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. khonkae- nensis 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 Coleop- tera 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 Multigene phylogeny and morphology of two new species 125 (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 um wide) than O. brunneipunctata (0.5 um), O. furcatosubulata (0.9 um), and O. houaynhangensis (0.5 um). 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. 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