Research Article
Research Article
Two new entomopathogenic species of Ophiocordyceps in Thailand
expand article infoYuan-Pin Xiao§, Sinang Hongsanan§|, Kevin D. Hyde§, Siraprapa Brooks§, Ning Xie|, Feng-Yao Long, Ting-Chi Wen
‡ Guizhou University, Guiyang, China
§ Mae Fah Luang University, Chiang Rai, Thailand
| Shenzhen University, Shenzhen, China
Open Access


Ophiocordyceps is entomopathogenic and the largest studied genus in the family Ophiocordycipitaceae. Many species in this genus have been reported from Thailand. The first new species introduced in this paper, Ophiocordyceps globiceps, differs from other species based on its smaller perithecia, shorter asci and secondary ascospores and additionally, in parasitising fly species. Phylogenetic analyses of combined LSU, SSU, ITS, TEF1α and RPB1 sequence data indicate that O. globiceps forms a distinct lineage within the genus Ophiocordyceps as a new species. The second new species, Ophiocordyceps sporangifera, is distinguished from closely related species by infecting larvae of insects (Coleoptera, Elateridae) and by producing white to brown sporangia, longer secondary synnemata and shorter primary and secondary phialides. We introduce O. sporangifera based on its significant morphological differences from other similar species, even though phylogenetic distinction is not well-supported.


2 new taxa, Hypocreales , morphology, phylogenetic, taxonomy


The genus Ophiocordyceps was introduced by Petch (1931) to accommodate species which have different features of asci and ascospores from Cordyceps (Petch 1931). Ophiocordyceps was treated as a subgenus of Cordyceps by Kobayasi (1941, 1982) and Mains (1958). Sung et al. (2007a) established the new family Ophiocordycipitaceae in Hypocreales (Sordariomycetes) and revised Ophiocordyceps as the type genus based on phylogenetic analyses. This is followed in the Outline of Ascomycetes (Wijayawardene et al. 2018). The main characters of the sexual morph species of Ophiocordyceps are fibrous, hard, pliant-to-wiry, dark stromata with superficial to immersed perithecia (Sung et al. 2007a, Ban et al. 2015). The asexual morphs in the majority of species have hirsutella-like and hymenostilbe-like features (Kepler et al. 2013, Maharachchikumbura et al. 2015, 2016). The hosts of species in Ophiocordyceps are larval lepidopterans and coleopterans, adult hymenopterans, hemipterans, dipterans, orthopterans or dragonflies (Odonata) and, in few cases, spiders (Kobayasi 1941, Mains 1958, Sung et al. 2007a, Ban et al. 2015). Hitherto, Ophiocordyceps included 233 species (Index Fungorum, June 2018) with a worldwide diversity (Sung et al. 2007a, Ban et al. 2015, Spatafora et al. 2015, Shrestha et al. 2017).

Thailand is located in the tropical areas with a rich biodiversity (Luangsa-ard et al. 2008, Aung et al. 2008, Luangsa-ard et al. 2010, Hyde et al. 2017, Hyde et al. 2018). A variety of entomopathogenic species (more than 400 species) (Index Fungorum, June 2018, Luangsa-ard et al. 2008, Luangsa-ard et al. 2010) were reported from Thailand after the first species recorded by Petch in 1932. In this study, we introduce two new species of Ophiocordyceps, which were found on larvae of insects (Lepidoptera, Cossidae) and adult Diptera. The descriptions of these two new species and phylogenetic evidence for the new taxa are provided. Morphological differences between two new species and their related species are also discussed.


Collection, isolation, and morphology study

Specimens were collected in The Mushroom Research Centre, Chiang Mai, Thailand, from soil and grass litter and taken to the laboratory. Fruiting bodies were examined using free hand sections under a stereomicroscope. Water-mounted slides were prepared for a microscope study and photographed under a compound microscope. Strains were isolated from single spores by using the protocol in Chomnunti et al. (2014). Cultures were incubated at 25 °C for 4–10 weeks on potato extract agar (PDA) in light-promoted sporulation.

DNA extraction, PCR amplification and determination of DNA sequences

DNA was extracted from both dried specimens and cultures by using E.Z.N.A.TM Fungal DNA MiniKit (Omega Biotech, CA, USA), according to the manufacturers protocols. Universal known primers were used in PCR amplification; ITS4/ITS5 for internal transcribed spacer gene region (ITS), NS1/NS4 for partial small subunit ribosomal RNA gene region (SSU), LROR/LR5 for partial large subunit rDNA gene region (LSU) (Vilgalys and Hester 1990, White et al. 1990), 983F/2218R for partial translation elongation factor 1-alpha gene region (TEF1α) (Sung et al. 2007b) and CRPB1A/RPB1Cr for partial RNA polymerase II largest subunit gene region (RPB1) (Castlebury et al. 2004). PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China. Specimen was performed by using TaKaRa PMD18-T vector system (TaKaRa Biotechnology, Dalian, China), while PCR products could not be sequenced directly.

Phylogenetic analyses

Sequence data were obtained from GenBank based on previous studies as listed in Table 1. MAFFT v.7 was used to align combined datasets of ITS, SSU, LSU, TEF1α and RPB1 regions (Katoh and Standley 2013, BioEdit (Hall 2011) was used to check alignment manually. Gaps were treated as missing data. Tolypocladium inflatum W. Gams and T. ophioglossoides (J.F. Gmel.) C.A. Quandt et al. (Kepler et al. 2012, Schoch et al. 2012) were selected as outgroup taxa.

Table 1.

Sources of isolates and GenBank accession numbers used in the paper.

Species Insecta Voucher SSU ITS LSU TEF1α RPB1 References
H. dipterigena Diptera NHJ12170.02 GU723771 GU797126 Luangsa-ard et al. 2011
O. acicularis Coleoptera (larva) OSC 110988 EF468951 EF468804 EF468745 EF468853 Sung et al. 2007a
O. agriotidis Coleoptera (larva) ARSEF 5692 DQ522540 JN049819 DQ518754 DQ522322 DQ522368 Ban et al. 2015
O. amazonica Orthoptera (Acrididae imago) Ophama2026 KJ917562 KJ917571 KM411989 KP212902 Sanjuan et al. 2015
O. annulata Coleoptera CEM 303 KJ878915 KJ878881 KJ878962 KJ878995 Quandt et al. 2014
O. aphodii Coleoptera ARSEF 5498 DQ522541 DQ518755 DQ522323 Spatafora et al. 2007
O. appendiculata Coleoptera (larva) NBRC 106960 JN941728 JN943326 JN941413 AB968577 JN992462 Ban et al. 2015
O. arborescens Cossida (larva) NBRC 105891 AB968398 AB968414 AB968572 Ban et al. 2015
O. australis Hymenoptera (ant) Ophaus992 KC610785 KC610766 KC610731 KF658663 Ban et al. 2015
O. barnesii Coleoptera (larva) BCC28560 EU408776 EU408773 Luangsa-ard et al. 2010
O. brunneinigra Hemiptera (Cicadellidae) TBRC 8093 MF614654 MF614638 MF614668 Luangsa-Ard et al. 2018
O. brunneiperitheciata Lepidoptera (larva) TBRC 8100 MF614658 MF614643 Luangsa-Ard et al. 2018
O. brunneipunctata Coleoptera (Elateridae larva) OSC 128576 DQ522542 DQ518756 DQ522324 DQ522369 Spatafora et al. 2007
O. buquetii Hymenoptera (Formicidae) HMAS 199613 KJ878939 KJ878904 KJ878984 KJ879019 Quandt et al. 2014
O. citrina Hemiptera TNS F18537 KJ878903 KJ878983 Quandt et al. 2014
O. clavata Coleoptera (larva) NBRC 106962 JN941726 JN943328 JN941415 AB968587 JN992460 Schoch et al. 2012
O. coccidiicola Insect NBRC 100682 AB968404 AB968419 AB968583 Ban et al. 2015
O. coccidiicola Insect HMAS199612 KJ878917 AB027377 KJ878884 KJ878965 KJ878998 Quandt et al. 2014
O. coenomyia Coenomyia (larva) NBRC 108993 AB968384 AB968396 AB968412 AB968570 Ban et al. 2015
O. communis Coleoptera NHJ 12581 EF468973 EF468831 EF468775 Quandt et al. 2014
O. cossidarum Lepidoptera (larva) MFLU 17-0752 MF398186 MF398187 MF928403 MF928404 Hyde et al. 2017
O. crinalis Lepidopteran (larva) HIMGD17327 EU149926 Zhang et al. 2007
O. curculionum Coleoptera (adult Curculionidae) OSC 151910 KJ878918 KJ878885 KJ878999 Quandt et al. 2014
O. cylindrospora Hymenoptera (adult wasp) MFLU: 17-1961 MG553651 MG553635 MG553652 Hyde et al. 2018
O. dipterigena Diptera (adult fly) MY621 GU723764 GU797126 Luangsa-ard et al. 2011
O. dipterigena Diptera (adult fly) MRCIF71 EU573346 Freire 2015
O. dipterigena Diptera (adult fly) OSC 151912 KJ878920 KJ878887 KJ878967 KJ879001 Quandt et al. 2014
O. elongata Lepidoptera (larva) OSC 110989 EF468808 EF468748 EF468856 Sung et al. 2007a
O. emeiensis Lepidoptera (larva) G96031 AJ309347 Liu et al. 2002
O. entomorrhiza Lepidoptera KEW 53484 EF468954 JN049850 EF468809 EF468749 EF468857 Quandt et al. 2014
O. evansii Hymenoptera (Pachycondylaharpax) Ophsp 858 KC610796 KC610770 KC610736 KP212916 Sanjuan et al. 2015
O. forquignonii Diptera (adult fly) OSC 151908 KJ878922 KJ878889 KJ879003 Quandt et al. 2014
O. formicarum Camponotus (Ant) BCMU CF 01 AB222678 Freire 2015
O. formicarum Camponotus (Ant) BCMU CF 02 AB222679 Freire 2015
O. formosana Coleoptera (larva) MFLU: 15-3888 Li et al. 2016
O. fulgoromorphila Hemiptera (Fulgoridae adult) Ophara717 KC610794 KC610760 KC610729 KF658676 Sanjuan et al. 2015
O. geometridicola Lepidoptera (Geometridae) TBRC 8095 MF614648 MF614632 MF614663 Luangsa-Ard et al. 2018
O. globiceps Diptera (adult fly) MFLUCC 18-0495 MH725811 MH725815 MH725829 MH727387 This study
O. globiceps Diptera (adult fly) MFLU 18-0661 MH725812 NH725816 MH725830 MH727388 This study
O. gracilis Lepidoptera (larva) EFCC 8572 EF468956 JN049851 EF468811 EF468751 EF468859 Kepler et al. 2012
O. hemisphaerica Diptera (adult fly) FLOR 59525 KX197233 Hyde et al. 2016
O. heteropoda Hemiptera (cicada nymph) OSC 106404 AY489690 AY489722 AY489617 AY489651 Castlebury et al. 2004
O. irangiensis Hymenoptera (adult ant) OSC 128579 EF469123 EF469076 EF469060 EF469089 Sung et al. 2007a
O. issidarum Hemiptera (adult) MFLU:17-0751 MF398185 MF398188 Hyde et al. 2017
O. karstii Hepialus (larva) MFLU:15-3884 KU854952 KU854945 KU854943 Li et al. 2016
O. konnoana Coleoptera (larva) EFCC 7315 EF468959 EF468753 EF468861 Sung et al. 2007a
O. lanpingensis Hepialus (larva) YHOS0707 KC417459 KC417461 KC417463 KC417465 Chen et al. 2013
O. lloydii Hymenoptera (Camponotus) OSC 151913 KJ878924 KJ878891 KJ878970 KJ879004 Quandt et al. 2014
O. longissima Hemiptera (cicada nymph) NBRC 108989 AB968394 AB968407 AB968421 AB968585 Sanjuan et al. 2015
O. macroacicularis lepidopterans (larvae) NBRC 105888 AB968389 AB968401 AB968417 AB968575 Ban et al. 2015
O. melolonthae Coleoptera (Scarabeidae larva) OSC 110993 DQ522548 DQ518762 DQ522331 DQ522376 Spatafora et al. 2007
O. multiperitheciata Lepidoptera (larva) BCC 69008 MF614657 MF614641 Luangsa-Ard et al. 2018
O. myrmecophila Hymenoptera (adult ant) MFLU 16-2912 MF351730 MF351726 MF372585 MF372759 Xiao et al. 2017
O. myrmicarum Formicidae (adult ant) ARSEF11864 KJ680150 JX566973 KJ680151 Simmons et al. 2015
O. neovolkiana Coleoptera OSC 151903 KJ878930 KJ878896 KJ878976 KJ879010 Quandt et al. 2014
O. nigra Hemiptera TNS 16252 KJ878941 KJ878906 KJ878986 Quandt et al. 2014
O. nigrella Lepidoptera (larva) EFCC 9247 EF468963 JN049853 EF468818 EF468758 EF468866 Sung et al. 2007a
O. nutans Hemiptera (Pentatomidae adult) OSC 110994 DQ522549 DQ518763 DQ522333 DQ522378 Spatafora et al. 2007
O. odonatae Odonata (Dragonfly) TNS F18563 D86055 AB104725 Ito and Hirano 1997
O. pauciovoperitheciata Lepidoptera (larva) TBRC 8106 MF614652 MF614633 Luangsa-Ard et al. 2018
O. pseudoacicularis Lepidoptera (larva) TBRC 8102 MF614646 MF614630 MF614661 Luangsa-Ard et al. 2018
O. pulvinata Hymenoptera (adult ant) TNS-F 30044 GU904208 GU904209 GU904210 Quandt et al. 2014
O. purpureostromata Coleoptera TNS F18430 KJ878931 KJ878897 KJ878977 KJ879011 Quandt et al. 2014
O. pseudolloydii Formicidae (adult ant) MFLU 15-1425 MF351725 MF372758 MF372761 Xiao et al. 2017
O. ramosissimum Lepidoptera (larva) GZUHHN8 KJ028012 KJ028007 KJ028014 KJ028017 Wen et al. 2014
O. ravenelii Coleoptera (larva) OSC 110995 DQ522550 DQ518764 DQ522334 DQ522379 Spatafora et al. 2007
O. rhizoidea Isoptera (adult termite) NHJ 12529 EF468969 EF468824 EF468765 EF468872 Sung et al. 2007a
O. robertsii Lepidoptera (Hepialidae larva) KEW 27083 EF468826 EF468766 Sung et al. 2007a
O. rubiginosiperitheciata Coleoptera (larva) NBRC 106966 JN941704 JN943344 JN941437 AB968582 JN992438 Ban et al. 2015
O. sinensis Lepidopteran pupa EFCC7287 EF468971 JN049854 F468767 EF468874 Sung et al. 2007a
O. sobolifera Hemiptera (cicada nymph) NBRC 106967 AB968395 AB968409 AB968422 AB968590 Ban et al. 2015
O. sp FMF147 KX197238 Freire 2015
O. sp OSC 110997 EF468976 EF468774 EF468879 Quandt et al. 2014
O. spataforae Hemiptera (Fulgoridae) NHJ 12525 EF469125 EF469078 EF469063 EF469092 Sung et al. 2007a
O. sphecocephala Hymenoptera (adult wasp) NBRC 101753 JN941695 JN943350 JN941446 AB968592 JN992429 Ban et al. 2015
O. sporangifera Lepidoptera (Cossidae) MFLUCC 18-0492 MH725814 MH725818 MH725832 MH727390 MH727392 This study
O. sporangifera Lepidoptera (Cossidae) MFLU 18-0658 MH725813 MH725817 MH725831 MH727389 MH727391 This study
O. stylophora Coleoptera (Elateridae larva) OSC 111000 DQ522552 JN049828 DQ518766 DQ522337 DQ522382 Spatafora et al. 2007
O. superficialis Insect MICH 36253 EF468983 EF468883 Sung et al. 2007a
O. thanathonensis Hymenotera (adult ant) MFU 16-29010 MF882926 MF850375 MF850375 MF872614 MF872616 Xiao et al. 2017
O. tricentri Hemiptera (Cercopidae) NBRC 106968 AB968393 AB968410 AB968423 AB968593 Ban et al. 2015
O. unilateralis Hymenoptera (Camponotus) OSC 128574 DQ522554 DQ518768 DQ522339 DQ522385 Spatafora et al. 2007
O. variabilis Diptera (larva) OSC 111003 EF468985 EF468839 EF468779 EF468885 Sung et al. 2007a
O. xuefengensis Lepidoptera (Hepialidae larva) GZUH2012HN19 KC631788 KC631803 KC631794 KC631799 Wen et al. 2013
O. yakusimensis Hemiptera (cicada nymph) HMAS 199604 KJ878938 KJ878902 KJ879018 Quandt et al. 2014
T. inflatum Coleoptera (larva) OSC 71235 EF469124 JN049844 EF469077 EF469061 EF469090 Kepler et al. 2012
T. ophioglossoides Fungi (Elaphomyces sp.) NBRC 106332 JN941732 JN943322 JN941409 JN992466 Schoch et al. 2012

Maximum likelihood trees (ML) were estimated by using the software RAxML 7.2.8 Black Box (Stamatakis 2006, Stamatakis et al. 2008) in the CIPRES Science Gateway platform (Miller et al. 2010). MrModeltest v.2.3 (Nylander 2004) was used to determine the best-fit model of evolution for Bayesian analyses. MrBayes v.3.1.2 (Ronquist and Huelsenbeck 2003) was used to evaluate posterior probabilities (PP) (Rannala and Yang 1996, Zhaxybayeva and Gogarten 2002) by Markov Chain Monte Carlo sampling (BMCMC). Six simultaneous Markov chains were run for 10,000,000 generations, trees were sampled every 100th generation and 100,001 trees were obtained. The first 25% of trees (25,000) were discarded, as they represented the burn-in phase of the analyses, while the remaining trees (75,001) were used for calculation of posterior probabilities in the majority rule consensus tree (critical values for the topological convergence diagnostic is 0.01). Trees were figured in FigTree v1.4.0 programme (Rambaut 2012). Bayesian Posterior Probabilities (BYPP) equal to or great than 0.90 were given below each node (Fig. 1).

Figure 1. 

Phylogram of Ophiocordyceps globiceps and O. sporangifera generated from maximum likelihood (RAxML) analysis of ITS, SSU, LSU, RPB1 and TEF1α sequence data. Tolypocladium inflatum and T. ophioglossoides were used as outgroup taxon. Maximum likelihood bootstrap values greater than 75% and Bayesian posterior probabilities over 0.90 were indicated above the nodes. The new species are indicated in red.


Molecular phylogeny

Eighty-seven taxa (including the four with new sequence data) were included in the combined ITS, SSU, LSU, RPB1 and TEF1α dataset (Table 1), which comprises 3894 characters with gaps; 1011 characters for SSU, 824 for LSU, 561 for ITS, 880 for TEF1α and 618 for RPB1. Tree topology of the RAxML analysis was similar to the Bayesian analysis. The best scoring RAxML tree with a final likelihood value of -46932.268101 is presented (Fig. 1). The matrix had 2081 distinct alignment patterns, with 35.22% of undetermined characters or gaps. Parameters for the GTR model of the concatenated dataset were as follows: Estimated base frequencies; A = 0.240006, C = 0.270755, G = 0.276725, T = 0.212514; substitution rates AC = 1.073676, AG = 3.611556, AT = 1.170890, CG = 1.176549, CT = 6.339087, GT = 1.000; gamma distribution shape parameter α = 0.265589.


Ophiocordyceps globiceps Y.P. Xiao, T.C. Wen & K.D. Hyde, sp. nov.

Fig. 2


The specific epithet refers to the feature of the secondary hemispherical to globoid fertile head.

Sexual morph: Stromata 4–8 mm long × 0.5–1 mm diam., one or several from the host, stipitate, capitate, unbranched, cinnamon to yellow. Stipe 3.5–7.5 mm long, 0.2–0.5 mm diam., yellow, cylindrical, with a fertile apex. Fertile head 1–1.5 mm long, 1–1.2 mm diam., cinnamon to yellow, single, hemispherical to globoid. Perithecia 538–663 × 182–247 μm (x̄= 600 × 214 µm, n = 60), immersed, ovoid to elongated pyriform, thick-walled, vertical with the ostioles opening on the upper surface of the head. Peridium 17–22 µm (x̄ = 20 µm, n = 90) wide, hyaline, of textura porrecta to textura prismatica to textura angularis. Asci 373–454 × 5.7–8.2 μm (x̄ = 413 × 7 µm, n = 90), 8-spored, hyaline, filiform, with a thick apex. Apical cap 4.4–6.4 × 4.9–5.7 μm (x̄ = 5.4 × 5.3 µm, n = 60), thick, with a small channel in the centre. Ascospores 240–303 × 1.8–2.3 μm (x̄ = 272 × 2.1 µm, n = 60), filiform, hyaline, multiseptate. Secondary ascospores 4–5.4 × 1.2–1.9 μm (x̄ = 4.7 × 1.6 µm, n = 90) cylindrical to fusoid, 1-celled, straight, hyaline, smooth-walled. Asexual morph: Undetermined.

Figure 2. 

Ophiocordyceps globiceps (holotype MFLU 18–0661). a Habitat b Ascostroma emerging from infected fly c Host d Fertile head of ascostroma e Vertical section of the stroma f Section of ascomata g Peridium h, i Asci k Apical cap of asci l, q Part of ascospore m, n Secondary ascospores o Upper side of the culture p Reverse side of the culture. Scale bars: 1000 µm (b–d), 500 µm (e, f), 100 µm (h, i), 20 µm (g), 10 µm (k, l), 5 µm (m, n, q), 5 cm (o, p).

Culture characteristics

growing on PDA, reaching 5 cm diam., after 6 weeks at 25 °C, superficial cottony, whitened, loose, reverse yellow. After 10 weeks at 25 °C, reaching 6 cm diam., no conidiogenous structures observed.

Material examined

THAILAND, Ranong, Tambon Khao Niwet, parasitise on fly (Muscidae, Diptera) 7 mm long, 3 mm wide, brown to dark brown, without hyphae on the surface, collected on the grass stem, 19 July 2015, YuanPin Xiao, (MFLU 18–0661, holotype, ex-type living culture, MFLUCC 18–0495); Chiang Mai, Thailand, on adult fly (Diptera), 6.5 mm long, 2.7 mm wide, brown to dark brown, without hyphae on the surface, collected on the grass, 19 July 2017, YuanPin Xiao, (MFLU 18–0662, paratypes, living culture MFLUCC 18–0496).


In the phylogenetic tree, Ophiocordyceps globiceps is closely related to O. dipterigena (Berk. & Broome) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafor. (Thailand) and O. hemisphaerica Mafalda-Freire, Reck & Drechsler-Santos (Brazil), which infect flies (Luangsa-ard et al. 2008, Hyde et al. 2016). Ophiocordyceps globiceps also groups with Ophiocordyceps sp. (FMF147) (106bp ITS differ), which was introduced by ITS sequence data and without any other detail (Freire 2015). Ophiocordyceps globiceps has 60 bp that differ from O. dipterigena (MY621, Thailand) in the ITS region, 19 bp in TEF1α. It has 87 bp that differ from Hymenostilbe dipterigena Petch (NHJ12170, Thailand, asexual morph of O. dipterigena) in the ITS region and 20 bp in TEF1α. Ophiocordyceps globiceps also has 94 bp (ITS) that differ from O. dipterigena (MRCIF71, Thailand), which only has ITS and without any details. Ophiocordyceps globiceps has 104 bp that differ from O. hemisphaerica (FLOR 59525) in the ITS region and has 21 bp in nrSSU, 97 bp in nrLSU, 74 bp in TEF1α that differ from O. dipterigena (OSC 151913).

We compared the new species with other Ophiocordyceps species which infect flies (Diptera) or are morphologically similar to O. globiceps (Table 2). Ophiocordyceps globiceps differs from three records of O. dipterigena found in Sri Lanka, Japan and Thailand by producing single smaller stroma, smaller and shorter perithecia, shorter asci and smaller ascospores (Table 2). Cordyceps sakishimensis Kobayasi & Shimizu, Ophiocordyceps discoideicapitata (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, Ophiocordyceps forquignonii (Quél.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, Ophiocordyceps hemisphaerica Mafalda-Freire, Reck & Drechsler-Santos, Ophiocordyceps lacrimoidis Mafalda-Freire, Reck & Drechsler-Santos and Cordyceps muscicola Möller (= Ophiocordyceps muscicola) have been reported as fly infected taxa (Saccardo 1891, Möller 1901, Kobayasi and Shimizu 1982, Freire 2015, Hyde et al. 2016), but their morphology is different from O. globiceps (see Table 2). Cordyceps sakishimensis is distinct from O. globiceps in having white, longer, cylindrical stromata and larger superficial perithecia. Ophiocordyceps discoideicapitata differs from O. globiceps by producing smaller stromata, pyriform, larger perithecia and longer part-spores (Table 2) (Kobayasi and Shimizu 1982). Ophiocordyceps forquignonii is distinct from O. globiceps in having a cylindrical fertile apex and oval secondary ascospores (Table 2) (Saccardo 1891). Molecular data indicate that the new species has 26 bp in nrSSU and 89 bp in nrLSU that are different from O. forquignonii. Ophiocordyceps hemisphaerica is different from O. globiceps in having longer stomata, larger obpyriform perithecia, longer asci and longer fusoid part-spores (Hyde et al. 2016). Ophiocordyceps lacrimoidis (Diptera infected species) was not considered in our phylogenetic sampling as the DNA (ITS) sequence did not align well with other species, but its DNA sequence differed by 154 bp in the ITS region from the sequence of O. globiceps. However, Ophiocordyceps lacrimoidis is morphologically different from our new species in producing longer stipe, obpyriform, slightly curved perithecia, longer asci and longer part spores. Cordyceps muscicola was revised as Ophiocordyceps muscicola by Freire (2015), while it is different from O. globiceps in having longer stromata, larger pyriform perithecia, longer asci and longer part-spores (Möller 1901, Freire 2015). We would like to introduce Ophiocordyceps globiceps as a new species based on the phylogenetic and morphological analyses.

Table 2.

Synopsis of Ophiocordyceps species discussed in the paper.

Species Location Host Stromata (mm) Stipe (mm) Fertile part (mm) Perithecia (μm) Asci (μm) Ascospores (μm) Part-spores (μm) Reference
C. sakishimensis Japan Diptera 6–7 long, cylindrical, white 500 × 250–260, superficial, ovoid 4–6 × 1, cylindrical Kobayasi and Shimizu 1983
O. dipterigena (First record) Sri Lanka 5–10 × 1, pale Cylindrical Globose 10 × 1.5 Berkeley and Broome 1873, Freire 2015
O. dipterigena Japan Diptera 5–8 long, 1–2 wide, 0.5–1 wide, orange-cinnamon or cinnamon-brown 0.2–0.5 thick, orange-cinnamon to light yellow Narrowly ovoid or conoid, 700–900 × 240–400, wall 15–25 thick 480–600 long Filiform, multiseptate 6–12 × 1–1.5, cylindric or fusoid fragments Kobayasi 1941
O. dipterigena Thailand Diptera 4–10 long, pale cream-yellow to orange-brown 1–1.5 high, 1.5–2.5 diam., terminal, disc-like to subglobose 800–1000 × 200–300, narrowly ovoid to obclavate 450–600 × 4–6, cylindrical Filiform, breaking up into 64 part-spore 6–12 × 1–1.5, cylindrical to fusiform Luangsa-ard et al. 2008
O. discoideicapitata Japan Diptera 2.5–3.5 × 0.7–1.2, two 3–4, discoid, laterally conical 620–700 × 200–250, pyriform 5–6 diam., filiform 6–9 × 1, cylindrical, truncated Kobayasi and Shimizu 1982
O. forquignonii Diptera 3-6 long, subfiliform, with a cylindrical apex Cylindrical Ellipsoid Oval, 8 Saccardo 1891
O. globiceps Thailand Diptera 4–8 long × 0.5–1 diam., unbranched, cinnamon to yellow, one or several from host 3.5–7.5 long, 0.2–0.5 diam., cinnamon to yellow, cylindrical, with a fertile apex 1–1.5 long, 1–1.2 diam., yellow, hemispherical to globoid 538–663 × 182–247, ovoid to elongated pyriform 373–454 × 5.7–8 240–303 × 1.8–2.3, filiform, hyaline, multiseptate 4–5.4 × 1.2–1.9, cylindrical to fusoid This study
O. hemisphaerica Brazil Diptera (Muscidae) 12–20 × 0.8–1, unbranched, brown to greyish-brown 11–19 long, 0.8–1 wide, cylindrical, with a fertile apex 1–1.2 long, 2–4 diam., hemispherical 780–860 × 220–290, Obpyriform, slightly curved 500–640 × 5–6 Filiform, more than 52 septa 7–10 × 1–1.5, cylindrical to unusually fusoid Hyde et al. 2016
O. lacrimoidis Brazil Diptera 4–5 × 1, two, simple 3–4 long, 1 wide, cylindrical, epidermal layer brown, medullar region white to cream 1.2 long, 1.8–2.2 diam., discoid, pale to dark yellowish 650–700 × 200–250, immersed, obpyriform, slightly curved 350–450 × 5, narrow cylindrical Filiform, as long as asci, hyaline, more than 56 septa 8–14 × 2, cylindrical, hyaline Hyde et al. 2016
O. muscicola = C. muscicola Brazil Diptera 9–13 × 0.5–1, two to six, rarely branched 2–4 × 1–1.2, discoid 850–920 × 230–300, pyriform 550–700 × 5, filiform 650–700 × 2, 64 part-spores 11–14 × 2, terminal cylindrical, intermediates fusoids 8–10 × 1–2 Möller 1901, Freire 2015

Ophiocordyceps sporangifera Y.P. Xiao, T.C. Wen & K.D. Hyde, sp. nov.

Figs 3, 4


The specific epithet refers to the feature of the sporangium-bearing.

Sexual morph: Unknown. Asexual morph: Primary synnema 9–18 cm high 1–2 mm diam., arising from the head region of the larva, branching into 2–5, cylindrical, brown to deep brown, with small white fertile head on the top, not smooth. Fertile head 500–2000 µm long, 400–1000 µm diam., globose to subglobose, capitulum, white to brown, arising from the apical end of primary synnema, mess of sporangium on the surface. Sporangium 78–121 µm diam. (x̄ = 100 µm, n = 60), spherical, arising from the apical end of primary synnema, white colour when immature, becoming brown to dark brown after maturity, consisting of thick-walled cells. Secondary synnemata 1092–1937 × 21–34 µm, (x ‒= 1515 × 27 µm, n = 60), laterally from the primary synnema, brown to white, cylindrical, not smooth. Hyphae 1.8–2.8 µm wide (x̄ = 2.3 µm, n = 60), irregularly multi-septate, brown, cylindrical, smooth or rough, sometimes particularly expand. Phialides 25–40 × 1.3–2.5 µm (x̄ = 33 × 1.9 µm, n = 60), hirsutella-like, hyaline, solitary, unbranched, narrow slender, smooth. Conidia 6.7–9.8 × 2.5–3.8 µm (x̄ = 8.3 × 3.2 µm, n = 60), 1 cell, hyaline, subglobose to reniform, bound in mucilaginous spheres. Mucilaginous spheres 10.5–12.9 × 6.4–8.7 µm (x̄ = 11.7 × 7.5 µm, n = 60), composed of 1–12 conidia, hyaline, at phialide apex.

Figure 3. 

Ophiocordyceps sporangifera (holotype MFLU 18–0658). a Habitat b Synnemata on host surface c Host d, e Synnemata f Fertile head of primary synnema g Sporangium h Secondary synnema i Sporangium j, k, q Part of secondary synnema l Phialides m Conidia bound by deliquescing mucilaginous material n–p Conidia. Scale bars: 1 cm (c, d), 1000 µm (e), 200 µm (f, h, q), 100 µm (g, i), 50 µm (j), 20 µm (k, l), 10 µm (m–p).

Culture Characteristics

growing on PDA, reaching 2 cm diam., after 4 weeks at 25 °C,with circular, dense mycelium on the surface. After 6 weeks, the colour of the colony gradually deepened from white to dark brown from the periphery to the centre, with complex fold as 4 circle rings, reverse white to yellow in colour, with ring. Synnemata was produced after 8 weeks. Most of the characters are the same as the fresh collection except phialides and mucilaginous spheres. Phialides 56–86 µm long (x̄ = 71 µm, n = 60), 3–5 µm wide at base (x̄ = 4 µm, n = 60), 1.4–2.2 µm at top (x̄ = 1.8 µm, n = 60), hirsutella-like, hyaline, solitary, unbranched, narrow slender, smooth, 1–4 septa, not observed on host. Mucilaginous spheres 10.5–15.9 × 8.2–14.7 µm (x̄ = 12.7 × 11.5 µm, n = 60), 1–4 conidia, hyaline to brown. Observation stopped after 10 weeks.

Material examined

THAILAND, Chiang Mai, The Mushroom Research Centre, on dead larva of Elateridae, Coleoptera, 6.5 cm long 0.38 cm diam., brown to dark brown, with thallus inside (larva), 18 July 2015, YuanPin Xiao, (MFLU 18–0658, holotype); THAILAND, Chiang Mai, The Mushroom Research Centre, on dead larva of Elateridae, Coleoptera, 5.8 cm long 0.4 cm diam., brown to dark brown, with thallus inside (larva), 22 August 2015, YuanPin Xiao, (MFLU 18–0659, paratypes, ex-type living culture, MFLUCC 18–0492); THAILAND, Chiang Mai, Samoeng on larva insect of Elateridae, Coleoptera, 5.5 cm long 0.32 cm diam., brown to dark brown, with thallus inside (larva), 18 June 2017, YuanPin Xiao, (MFLU 18–0660, paratypes, living culture, MFLUCC 18–0493, MFLUCC 18–0494).


Ophiocordyceps sporangifera is closely related to O. myrmicarum D.R. Simmons & Groden in our phylogenetic tree (Fig. 1). The morphology of O. sporangifera is different from O. myrmicarum in having longer primary and secondary synnemata, a white to brown sporangium, shorter phialides and it infects insect larvae (Lepidoptera, Cossidae), while O. myrmicarum was found on an ant (Myrmica rubra) (Simmons et al. 2015). The phylogenetic analysis does not have good support, but O. sporangifera is distinct from O. myrmicarum. In the phylogenetic tree, the relationships of O. sporangifera and O. myrmicarum are obscure because they share one clade with short branch length (100% ML/ 1 BYPP), while the two strains of O. sporangifera clustered together with a low bootstrap support (88% ML/ 0.90 BYPP). The type strain of O. sporangifera has 0 bp in nrSSU, 3 bp in TEF1α and 5 bp in RPB1 that are different from O. myrmicarum. However, the morphological features of those two species are different, thus, they should be treated as two separate species (Table 3).

Figure 4. 

Ophiocordyceps sporangifera (culture) MFLUCC 18–0492. a Upper side of the culture b Reverse side of the culture c, d Synnemata growing on PDA medium e, g Synnemata f Mycelium h–j Phialides k Conidia l–n Conidia form mucilaginous spheres. Scale bars: 1 cm (a, b), 5000 µm (c), 1000 µm (d), 500 µm (e), 100 µm (f, g), 50 µm (h–j), 10 µm (k–n).

Table 3.

Synopsis of Ophiocordyceps species discussed in the paper.

Species Ophiocordyceps myrmicarum Ophiocordyceps sporangifera
Host Myrmica rubra (Hymenoptera) Elateridae, Coleoptera
Primary synnemata Whitish-yellow aging to rufous brown 9–18 cm high 1–2 mm diam., brown to deep brown
Secondary synnemata (µm) Hyaline aging to rufous brown, up to 350 long, narrow (25) at base, common on agar but not observed on host Brown to white, not smooth 1092–1937 × 21–34, arising from the all parts of the primary synnemata, observed on both of the host and agar
Primary phialides (µm) Subulate, hyaline or pigmented at base, 39.9–86.2 long, 3.6–5.4 wide at base Slender, solitary, hyaline, unbranched, narrow, smooth, 25–40 × 1.3–2.5
Secondary phialides (µm) Subulate, 27.2–47.0 long, 2.4–3.3 wide at base Narrow slender, 56–86 long, 3–5 wide at base, 1.4–2.2 at top, 1–4 septa, common on culture but not observed on host
Sporangium (µm) No observed 78–121 diam., spherical, white immature, brown after mature
Conidia (µm) 7.3–9.6 × 3.2–5.1 reniform to ovoid, bi-guttulate, aseptate 6.7–9.8 × 2.5–3.8, subglobose to reniform
Mucilaginous spheres (µm) Composed of 1–4 conidia, hyaline to brown, at phialide apex 10.5–12.9 × 6.4–8.7, composed of 1–12 conidia, hyaline on host, 1–4 conidia on culture, hyaline to brown on culture
Reference Simmons et al. 2015 This study


We introduce two new entomopathogenic species of Ophiocordyceps, one from Coleoptera (Elateridae) and the other from flies (Diptera). Morphological and phylogenetic analyses have provided insights to resolve generic delimitation (Sung et al. 2007a, Jeewon and Hyde 2016). Most of the species of this genus are parasitic on insects (Sung et al. 2007a, Maharachchikumbura et al. 2015, Wijayawardene et al. 2017). The sexual morph species in this genus is characterised by fibrous, hard, pliant-to-wiry, dark-coloured stroma with superficial to immersed perithecia (Sung et al. 2007a, Ban et al. 2015, Maharachchikumbura et al. 2015), while the asexual morph species have mainly hymenostilbe-like and hirsutella-like features, branched or unbranched phialides with oval to fusiform conidia (Kepler et al. 2013, Maharachchikumbura et al. 2015, 2016).

Ophiocordyceps globiceps groups with H. dipterigena, O. dipterigena, Ophiocordyceps sp. and O. hemisphaerica in the phylogenetic tree with high bootstrap support, while four of these species are reported as fly (Diptera) parasitic fungi (Kobayasi 1941, Saccardo 1891, Luangsa-ard et al. 2011, Hyde et al. 2016). Ophiocordyceps globiceps differs from closely related species by producing capitate, stipitate ascostromata, vertical, narrowly ovoid to obclavate, occasionally irregular perithecia and cylindrical secondary ascospores. Both morphology and phylogenetic analyses clearly show O. globiceps as a new species within Ophiocordyceps.

Ophiocordyceps sporangifera is an asexual morph species and groups with O. myrmicarum in the phylogenetic tree (Fig. 1). Ophiocordyceps sporangifera can be distinguished from O. myrmicarum by infecting and parasitising larvae of insects (Lepidoptera, Cossidae), producing white to brown sporangium, longer primary and secondary synnemata and shorter primary and secondary phialides. The new species can be defined based on the distinctive morphological characters even through the phylogenies are not well-supported (Jeewon and Hyde 2016). In case of intricate differences between a gene tree and a species tree and, in addition, several morphs can be under the influence of many genes which are not really being reflected in the phylogeny (Jeewon and Hyde 2016). In our study, morphological characters strongly support O. sporangifera as a new species within Ophiocordyceps, even through phylogenetic analysis is not well-resolved. In this case, other loci which have more phylogenetic variation than the current loci may be able to differentiate these two species.


This work was jointly supported by the National Natural Science Foundation of China (No. 31460012 & 31760014) and the Science and Technology Foundation of Guizhou Province (No. [2016]2863 & [2017]5788). Yuanpin Xiao also thanks the future of specialist fungi in a changing climate: baseline data for generalist and specialist fungi associated with ants, Rhododendron species and Dracaena species (grant no: DBG6080013), Impact of climate change on fungal diversity and biogeography in the Greater Mekong Subregion (grant no: RDG6130001) and the Mushroom Research Foundation, Chiang Rai, Thailand for supporting this research.


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