Morphological and genetic characteristics of the novel entomopathogenic fungus Ophiocordyceps langbianensis (Ophiocordycipitaceae, Hypocreales) from Lang Biang Biosphere Reserve, Vietnam

Abstract

An entomopathogenic fungus newly named Ophiocordyceps langbianensis was collected from Lang Biang Biosphere Reserve, located in Lam Dong Province, Vietnam. It is characterized as a species of Ophiocordyceps ( Ophiocordycipitaceae , Hypocreales ) having the unique characteristics of a cylindrical fertile part and several branched apical appendices. Each ascospore develops as two swollen, constricted part-spores. A phylogenetic analysis of multiple genes, including nrLSU , nrSSU , Rpb1 , ITS and Tef, supported its systematic position in the genus of Ophiocordyceps ; it is related to O. brunneipunctata . Based on morphological and phylogenetic analyses, O. langbianensis was confirmed as a new species from Vietnam.

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Morphological and genetic
characteristics of the novel
entomopathogenic fungus
Ophiocordyceps langbianensis
(Ophiocordycipitaceae,
Hypocreales) from Lang Biang
Biosphere Reserve, Vietnam
Thuan Duc Lao1, Thuy Ai Huyen Le1 & Nguyen Binh Truong2*
An entomopathogenic fungus newly named Ophiocordyceps langbianensis was collected from Lang
Biang Biosphere Reserve, located in Lam Dong Province, Vietnam. It is characterized as a species of
Ophiocordyceps (Ophiocordycipitaceae, Hypocreales) having the unique characteristics of a cylindrical
fertile part and several branched apical appendices. Each ascospore develops as two swollen,
constricted part-spores. A phylogenetic analysis of multiple genes, including nrLSU, nrSSU, Rpb1,
ITS and Tef, supported its systematic position in the genus of Ophiocordyceps; it is related to O.
brunneipunctata. Based on morphological and phylogenetic analyses, O. langbianensis was conrmed
as a new species from Vietnam.
e genus Ophiocordyceps, rst established by Petch in 1931, belongs to the family Ophiocordycipitaceae, order
Hypocreales, comprising approximately 250 species1,2. Originally, Ophiocordyceps was classied as a subgenus
of Cordyceps by Kobayasi (1941, 1982) and Mains (1958)3–5. In 2007, Sung etal. established a new called fam-
ily Ophiocordycipitaceae, comprising Ophiocordyceps, based on morphological and phylogenetic analyses6,7.
e distinction of the genus Ophiocordyceps from Cordyceps was done due to the darkly pigmented stromata
of Ophiocordyceps, which are pliant, wiry or brous and tough in texture, compared to the brightly pigmented
stromata of Cordyceps7. Species of Ophiocordyceps are entomopathogenic on a wide range of insects. e hosts of
species of Ophiocordyceps are the larvas of Coleoptera and Lepidoptera as well as the adults of Araneae, Diptera,
Hemiptera, Hymenoptera, Odonata and Orthoptera3–7. Although Ophiocordyceps has worldwide distribution, the
tropics and subtropics are where the highest numbers of the species are recorded. Moreover, it is considered that
there is an underestimation of the number of Ophicordyceps species.
Vietnam is located in a tropical region with terrestrial ecosystems. e forests feature a rich biodiversity of
both ora and fauna due to the tropical monsoon climate with high temperature and rainfall. is is a favorable
environment for the development of entomopathogenic fungi. Lang Biang Biosphere Reserve is located in Lam
Dong Province and comprises a vast primitive jungle with the Lang Bian Mountain at its core, one of Vietnam’s
four biodiversity centers. During our expedition to discover the diversity of entomopathogenic fungi, we col-
lected the sample DL0017. In this study, we introduce this specimen as a new species of Ophiocordyceps that
parasitizes the larva of Coleoptera. We present a morphological description and phylogenetic analysis based on
the phylogenetic construction of nuclear large ribosomal subunit (nrLSU), nuclear small ribosomal subunit
(nrSSU) and RNA Polymerase II Subunit B1(rpb1) of species of Ophiocordyceps, including this new species.
OPEN
Faculty of Biotechnology, Ho Chi Minh City Open University, Ho Chi Minh City, Vietnam. Faculty of Biology, Dalat
University, Dalat, Lam Dong, Vietnam. *email: nguyentb@dlu.edu.vn
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Materials and methods
Fungal specimen collection. e specimen, DL0017, used for this study was collected from Lang Biang
Biosphere Reserve (N 12°2′19.0″, E108°26′04.7″, elevation 1680m) in 9th August, 2016. e specimen, includ-
ing the host, was extracted carefully, noted, and photographed in the eld using a digital camera. e specimen
was immediately wrapped in wax paper, placed in a collection bag, and taken to the laboratory.
Cultivation techniques. According to the identication of conidia, phialides and colony coloration, the
isolate cultures were grown on YMG media, composed of 4g/l yeast extract (Sigma-Aldrich, Germany), 10g/l
malt extract (Sigma-Aldrich, Germany), 4g/l glucose (Sigma-Aldrich, Germany), and incubated at 20°C for a
period of 20days with PDA media (potato extract 4g/l, dextrose 20g/l, agar 15g/l; Merck, Germany).
For fruit body induction, cultures were grown on millet substrate (millet/silkworm pupae powder = 20:1
(w/w)) and brown rice substrate (brown rice/silkworm pupae powder = 20:1 (w/w)) at 20°C under 12h light
and 12h darkness with relative humidity of over 90%.
Morphological study: macro- and micro-morphological analysis. Morphological observations
were carried out and recorded according to the guidelines of Kobayasi and Sung etal.3,4,7. e macroscopic
characteristics of the fresh fruit body were carefully observed, including the stipe, stroma, etc. Moreover, the
color was noted according to Kornerup and Wanscher8. Additionally, the host insect was identied based on
morphological characteristics,such as mandibulate mouthparts, antennae, shape of head and thorax. For the
micro-morphological analysis, one or two perithecia were removed from the stroma and placed on a microscope
slide in lactophenol-cotton blue to measure the sizes and shapes of the perithecia, asci and ascospores. Finally,
the nomenclatural novelty and descriptions were deposited in MycoBank.
DNA extraction, PCR amplication, target gene sequencing. Genomic DNA was isolated by using
the phenol/chloroform method (pH = 8)11. e fruiting body was incubated in a lysis buer (2.0% SDS, Tris–HCl
pH 8.0, 150mM NaCl, 10mM EDTA, 0.1mg/ml Proteinase K) at 65°C overnight. e supernatant was collected
by centrifugation, and a volume of 700 μL of phenol/chloroform/isoamyl alcohol (25:24:1) was supplemented
and centrifuged. e supernatant was collected and precipitated with absolute isopropanol. Finally, the isolated
genomic DNA was stored in Tris–EDTA buer at −20°C for further studies.
e primer pairs used to amplify nrLSU, nrSSU, rpb1, ITS and Tef regions are shown in Table1. e nal
volume of PCR was done in a total of 15 μL with the thermal program: 1 cycle at 95°C for 5min, 40 cycles at
95°C for 30s, X°C for 30s, 72°C for 2min, 1 cycle at 72°C for 5min (Note: X°C is the annealing tempera-
tures for each target gene shown in Table1); 5 μL aliquots of amplication product were electrophoresed on a
2.0% agarose gel and visualized in a UV transilluminator. e amplied product was sequenced at Nam Khoa
(Vietnam) company.
Taxa and nrLSU, nrSSU, rpb1, ITS and tef sequences collection, DNA proofreading and phy-
logeny analysis. e data set of nrLSU, nrSSU, rpb1, ITS and tef sequences were established by sequences
downloaded from Genbank (NCBI) and based on the previous data published by Sung etal.7. e nrLSU, nrSSU,
rpb1, ITS and tef were noted with accession number, name of taxon and locality. e amplied DNA sequences
were proofread to remove ambiguous signals at both ends by dierent soware, including Seaview 4.2.12 and
Chromas Lite 2.1.1. e phylogenetic tree was constructed based on neighbor-joining (NJ), maximum parsi-
mony (MP), and maximum likelihood (ML), using Molecular Evolutionary Genetics Analysis (MEGA) version
5. Additionally, the best evolution model was predicted using jModelTest.
Results
Taxonomy. Ophiocordyceps langbianensis T. D. Lao, T. A. H. Le & N. B. Truong, sp. nov.
Mycobank MB836716 Figs.1, 2, 3.
Table 1. e primers’ sequence used in this study. F: Forward primer; R: Reverse primer; Ta: Annealing
temperature.
Target gene Primer Sequence (5′–3′)Ta (oC) References
nrLSU LR0R (F) GTA CCC GCT GAA CTT AAG C 55 9
LR5 (R) ATC CTG AGG GAA ACTTC
nrSSU NS1 (F) GTA GTC ATA TGC TTG TCT C 42.2 10
NS4 (R) CTT CCG TCA ATT CCT TTA AG
Rpb1 CRPB1 (F) CCW GGY TTY ATC AAG AAR GT 55 6
RPB1Cr (R) CCNGCDATNTCR TTR TCC ATR TA
ITS ITS1F (F) CTT GGT CAT TTA GAG GAA GTAA 55 10
ITS4 (R) TCC TCC GCT TAT TGA TAT GC
Tef 983F (F) GCY CCY GGHCAY CGT GAY TTY AT 55 10
2218R (R) ATG ACA CCR ACR GCR ACR GTYTG
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Figure1. Overview of Ophiocordyceps langbianensis. (A–D) Ecology of collected plots; (E) Stroma developing
from the head of hosts; (F) Immature stromata of fungus emerging from the larva of Coleoptera; (G) Stromata
in moist soil surrounded by dried leaves.
Figure2. Ophiocordyceps langbianensis. (A) Stroma on host; (B–D) Fertile part and apical appendix, surface
of fertile part with perithecium ostioles, cortex; (E) Host; (F) Mycelium on the host; (G) Perithecia; (H, I) Asci
with thick cap; (J, K) Ascospores.
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Typication. VIETNAM. Lam Dong Province, Lang Bian Biosphere Reserve, Lang Bian mountain: N
12°02′19.0″, E108°26′04.7″; elevation 1680 m; humidity: over 85%; temperature: day 20 °C–22 °C, night:
14°C–16°C; collected between 9h00–15h00 of the day on 9 August, 2016, from the larva of a beetle of Coleoptera
in moist soil surrounded by dried leaves. Truong B.N. DL0017 (Holotype DLU; Iso VNMN, DLU).
Distribution. Vietnam, only known from Lang Bian Mountain.
Etymology. “Langbianensis” refers to Lang Bian Mountain, Lam Dong province, Vietnam.
Host. On the larva of a beetle of Coleoptera. Larva: 28–32mm long, hard-body, shiny, smooth, dark brownish
yellow; body composed of 13 segments with black edges; larva with three pairsof jointedlegs attached to thorax.
Habitat. Individuals of associated species appeared at thetype locality, including pioneer species such as Acer
laurinum (Aceraceae), Baccaurea harmandii (Euphorbiaceae), Castanopsis chinensis (Fagaceae), Eriobotrya poil-
anei (Rosaceae), Jasminum longisepalum (Oleaceae), Phoebe petelotii (Lauraceae) and Tetrastigma lanceolarium
(Vitaceae).
Sexual morph. Stroma arising from the head of the host larva, solitary, rarely branched, 40–100mm long;
host covered with thin, tough layer of mycelium. Stipe liform, cylindrical, 30–67mm × 0.7–1.0mm, pale yel-
low. Fertile portion, cylindrical, 7.0–14.0mm × 1.5–2.0mm, brownish yellow with dark brown ostiolar dots of
perithecia. Apical appendices, pale yellow, 2–10 primary or secondary branches, 4.0–10.0 × 0.5 mm. Perithe-
cia immersed, ovate or pyriform, 260–400μm × 100–190µm. Asci, cylindrical, 200–250μm × 5.0–6.0μm, with
thickened cap. Ascospores liform, multiseptate, articulated in long-chain aer discharging, sometimes break-
ing into 1-celled part spores, cylindrical, swollen, two waist-like constrictions, 5–7.5μm × 1.3–2µm.
Asexual morph. Germination of ascospores aer 48h on PDA; white colony, slow growingon YMG and PDA
media, 25.00mm and 24.58mm aer 40days (respectively); septate hyphae, branched, chlamydospores devel-
oping in intercalary or terminal cells. Aerial hyphae with divergent phialides; elliptical conidia in chains aer
release from phialide. Stromata without fertile part forming on cereal substrates. Minor dierences in morpho-
logical characteristics of stromata developing from dierent substrates. Stromata, white, branched when devel-
oping on millet substrate; brownish yellow, solitary, rarely branched, when developing on brown rice substrate.
Amplication of nrLSU, nrSSU, rpb1, ITS and tef genes. Target genes, including nrLSU, nrSSU, rpb1,
ITS and tef, were successfully amplied with corresponding primers (Table1). e bands of 950-bp, 1102-bp,
803-bps, 700-bps, and 1030-bps corresponding to the amplied nrLSU, nrSSU, rpb1, ITS and tef were observed
Figure3. Asexual states. (A) Ascospores germinating aer 96h; (B) Septate hyphae, branched, conidia in
intercalary or terminal cells; (C) White colony aer 45days on YMG media; (D) Aerial hyphae with divergent
phialides; (E) Elliptical conidia in chains aer release from the phialide, (F, G) Stromata growing on cereal
substrates.
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in the electrophoresis on 2.0% agarose gel. e PCR products were sequenced with the signal of the peaks in
both strands of target genes; the sequence was signicant, unique and good for reading.
The systematic concatenated nrLSU, nrSSU, rpb1, ITS and tef gene dataset. To construct a
phylogeny of major lineages, representative taxa were chosen based on previous study7. e data set of nrLSU,
nrSSU, rpb1, ITS and tef consisted of 50, 50, 46, 39 and 42 taxa representing the morphological and ecological
diversity of genera in Ophiocordycipitaceae, Clavicipitaceae, and Cordycipitaceae, including the outgroup taxon
Glomerella cingulata (Glomerellaceae, Glomerellales) (Table2). A combined concatenated dataset consisting of
30 representative taxa was constructed based on the list of individual target genes.
Molecular phylogeny analysis. e sequences of nrLSU, nrSSU, rpb1, ITS and tef of DL0017 were simi-
lar to the representative sequence of Cordyceps brunneipunctata (similarity > 90%), with accession numbers of
DQ518756, DQ522542, DQ522369, GU723777 and DQ522324. Sequences were aligned and edited using the
MEGA 5.2. Gaps were excluded from the phylogenetic analysis. e dataset of representative taxa and DL0017
target gene sequence consisted of 451bp for nrLSU, 674bp for nrSSU, 392bp for Rpb1, 158bp for ITS and 790bp
for tef. e evolution model that was most xed with nrLSU, nrSSU, Rpb1, ITS and tef were TN93 + G, K2 + G + I,
T92 + G + I, K2 + G, and TN93 + G + I respectively. e phylogenetic trees were generated with Neighbor Joining
(NJ), Maximum Parsimony (MP), and Maximum Likelihood (ML) methods with replication of 1000. Based on
the NJ, MP, and ML phylogenetic trees, individual nrLSU, nrSSU, Rpb1, ITS, and tef of DL0017 clustered together
with Ophiocordyceps brunneipunctata within separate branches with credible bootstrap (≥ 50%), suggesting that
these species are related (Table3).
Information from molecular phylogenetic analysis based on separate genes is not enough to reconstruct trees
for higher classication compared to multigene analysis. erefore, a combined data set, including 2,319bp of
ve target genes, nrLSU-nrSSU-Rpb1-ITS-tef, was analyzed. e evolution model that was most xed with the
combined dataset was TN93 + G + I, as determined by MEGA 5.2. e phylogenetic trees, based on analysis
of the combined data, could be broadly separated into three groups, which corresponded to the families of
Clavicipitaceae, Ophiocordycipitaceae and Cordycipitaceae. In the phylogenetic tree, DL0017 clustered with
Ophiocordyceps brunneipunctata with bootstraps of 100/100/100 (NJ/MP/ML phylogenetic tree) and formed a
separate, monophyletic branch. Within this monophyletic branch, DL0017 and O. brunneipunctata clustered
together closely, suggesting that these species were truly associated (Fig.4). e molecular phylogenetic analysis
conrmed that there were dierences between DL0017 and other related species.
To conrm the authenticity of DL0017 as the most closely associated with Ophiocordyceps brunneipunctata,
the reconstruction of Neighbor-Net network of DL0017 and its allies was performed. e Neighbor-Network
analysis supported the results from the phylogenetic analysis (Fig.5). e network presented three complex
groups, corresponding to three families: Clavicipitaceae, Ophiocordycipitaceae and Cordycipitaceae. e DL0017
closely clustered with Ophiocordyceps complex. Additionally, speciation was observed between the cluster of
DL0017 and O. brunneipunctata.
Comparison of Ophiocordyceps langbianensis with close species. In the phylogenetic analysis, the
Ophiocordyceps langbianensis clustered with Ophiocordyceps brunneipunctata with high bootstrap support, sug-
gesting a close relationship. To conrm the authenticity of DL0017 as a new species, we compared DL0017 and
its close species, O. brunneipunctata. It diered from O. brunneipunctata by the morphological characteristics
described in Table4. erefore, DL0017 was conrmed as a new species, namely O. langbianensis.
Discussion
Lang Biang Biosphere Reserve, located in Lam Dong Province, is classied as Vietnam’s biodiversity center and
considered a hotspot of fungal biodiversity, including entomopathogenic fungi. During our expedition to validate
the diversity of entomopathogenic fungi in Lang Biang Biosphere Reserve, the sample DL0017 was collected.
Morphological analysis indicated that DL0017, named Ophiocordyceps langbianensis, is a new taxon. Species
belonging to the family Ophicordycipitaceae have stromata that are darkly pigmented or rarely brightly colored),
tough, brous, pliant, and rarely eshy. Additionally, asci are usually cylindrical with thickened ascus apex.
Ascospores are usually cylindrical, multiseptate, and disarticulate into part-spores or non-disarticulating7. Our
specimen shares these common characteristics.
Based on the phylogenetic analysis, the specimen DL0017 clustered with Ophiocordyceps brunneipunctata in
Ophiocordycipitaceae12. However, the morphologies of these two species are dierent in many characteristics,
including color, size of stroma, stipe, and dots in the fertile portion. e apical appendix of O. brunneipunctata
lacks branching, while O. langbianensis has 2–10 branches. Additionally, the ascospores of O. brunneipunctata
break into part-spores, while the ascospores of O. langbianensis stick together to form a multiseptate chain,
separating into unicellular part-spores under a strong interaction force. Multiple gene sequences of the related
Ophiocordyceps species were used in the phylogenetic analysis. A comparison was done among the species listed
in Table4 with respect to cylindrical fertile portion, embedded perithecia, and an apical appendix. Among them,
only species of Cordyceps furcicaodata have a branch-forming apical appendix. In the comparison between
Cordyceps furcicaodata and O. langbianensis, Cordyceps furcicaodata was found to be smaller than O. langbian-
ensis. e stroma of Cordyceps furcicaodata arose from the middle of the host larva, while that of O. langbianensis
arose from one end of the insect larva13. As mentioned above, ascospores of O. langbianensis stick together to
form a multiseptate chain, which could only be ruptured into unicellular part-spores by a strong force, while
ascospores of Cordyceps furcicaodata oen break into unicellular part-spores.
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Table 2. Representative taxa information and GenBank accession numbers for sequences used in current
study. –: no accession number recorded. a Outgroup.
Tax on Genus nrLSU nrSSU rpb1 ITS Tef
Claviceps fusiformis Claviceps U17402 DQ522539 DQ522366 JN049817 DQ522320
Claviceps paspali Claviceps U47826 U32401 DQ522367 JN049818 DQ522321
Claviceps purpurea Claviceps AF543789 AF543765 AY489648 KJ529004 AF543778
Claviceps purpurea Claviceps EF469075 EF469122 EF469087 KX977396 EF469058
Metacordyceps chlamydosporia Metacordyceps DQ518758 DQ522544 DQ522372 – EF469069
Metaccordyceps taii Metacordyceps AF543787 AF543763 DQ522383 – AF543775
Metacordyceps liangshanensis Metacordyceps EF468815 EF468962 – – EF468756
Metacordyceps liangshanensis Metacordyceps EF468814 EF468961 – – EF468755
Conoideocrella luteorostrata Conoideocrella EF468850 EF468995 EF468906 JN049859 EF468801
Conoideocrella luteorostrata Conoideocrella EF468849 EF468994 EF468905 JN049860 EF468800
Ophiocordyceps acicularis Ophiocordyceps EF468805 EF468950 EF468852 JN049820 EF468744
Ophiocordyceps acicularis Ophiocordyceps EF468804 EF468951 EF468853 GU723772 EF468745
Ophiocordyceps apholli Ophiocordyceps DQ518755 DQ522541 – – –
Ophiocordyceps brunneipunctata Ophiocordyceps DQ518756 DQ522542 DQ522369 GU723777 DQ522324
Ophiocordyceps sinensis Ophiocordyceps EF468827 MF403011 EF468874 JN049854 EF468767
Ophiocordyceps stylophora Ophiocordyceps EF468837 EF468982 EF468882 – EF468777
Ophiocordyceps stylophora Ophiocordyceps DQ518766 DQ522552 DQ522382 JN049828 DQ522337
Ophiocordyceps australis Ophiocordyceps DQ518768 DQ522554 DQ522385 – –
Ophiocordyceps variabilis Ophiocordyceps EF468839 EF468985 EF468885 – EF468779
Ophiocordyceps entomorrhiza Ophiocordyceps EF468809 EF468954 EF468857 JN049850 EF468749
Ophiocordyceps gracilis Ophiocordyceps EF468810 EF468955 EF468858 AJ786563 EF468750
Ophiocordyceps gracilis Ophiocordyceps EF468811 EF468956 EF468859 AJ786564 EF468751
Ophiocordyceps heteropoda Ophiocordyceps AY489722 AY489690 AY489651 FJ765028 AY489617
Ophiocordyceps heteropoda Ophiocordyceps EF468812 EF468957 EF468860 JN049852 EF468752
Ophiocordyceps nigrella Ophiocordyceps EF468818 EF468963 EF468866 JN049853 EF468758
Ophiocordyceps rhizoidea Ophiocordyceps EF468825 EF468970 EF468873 JN049857 EF468764
Ophiocordyceps rhizoidea Ophiocordyceps EF468824 EF468969 EF468872 MH754720 EF468765
Beauveria caledonica Beauveria AF339520 AF339570 EF469086 HQ880817 EF469057
Cordyceps cf. pruinosa Cordyceps EF468820 EF468965 EF468868 – DQ522351
Cordyceps cf.pruinosa Cordyceps EF468821 EF468966 EF468869 – –
Cordyceps cf.pruinosa Cordyceps EF468823 EF468968 EF468871 – EF468761
Cordyceps cicadae Cordyceps MH879588 MH879636 MH885438 MH93774 –
Cordyceps cicadae Cordyceps MK761212 MK761207 MF416653 MH937742 –
Cordyceps kyusyuensis Cordyceps EF468813 EF468960 EF468863 – –
Cordyceps militaris Cordyceps AY184966 AY184977 DQ522377 – DQ522332
Cordyceps pruinosa Cordyceps AY184968 AY184979 DQ522397 – EF468763
Cordyceps scarabaeicola Cordyceps AF339524 AF339574 DQ522380 JN049827 DQ522335
Cordyceps staphylinidicola Beauveria EF468836 EF468981 EF468881 – EF468776
Lecanicillium antillanum Lecanicillium AF339536 AF339585 DQ522396 MH861888 DQ522350
Lecanicillium fusisporum Lecanicillium AF339549 AF339598 EF468889 – EF468776
Lecanicillium psalliotae Lecanicillium AF339559 AF339608 EF468890 – –
Lecanicillium tenuipes Lecanicillium AF339526 AF339576 DQ522387 JN036556 DQ522341
Cordyceps ninchukispora Cordyceps EF468846 EF468991 EF468900 – EF468795
Cordyceps ninchukispora Cordyceps EF468847 EF468992 EF468901 – EF468794
Simplicillium lamellicola Simplicillium AF339552 AF339601 DQ522404 MH854806 DQ522356
Simplicillium lanosoniveum Simplicillium AF339554 AF339603 DQ522405 –
Simplicillium lanosoniveum Simplicillium AF339553 AF339602 DQ522406 – DQ522357
Simplicillium obclavatum Simplicillium AF339517 AF339567 – MH860859 DQ522358
Glomerella cingulateaColletotrichum AF543786 AF543762 AY489659 FJ904831 AF543773
Glomerella cingulateaColletotrichum U48428 U48427 DQ858454 EU520087 AF543772
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e asexual morph of O. langbianensis consists of long and divergent phialides, elliptical conidia usually in
chains considered paecilomyces-like or purpureocillium-like14,15. Conversely, O. bruneipunctata produced a
mononematous hirsutella-like asexual morph from colonies aer 3–4weeks.
Conclusion
We successfully applied morphological characterization in combination with phylogenetic analysis of multiple
genes, including nrLSU, nrSSU, rpb1, ITS, and Tef, to delimit sample DL0017, collected from Lang Biang Bio-
sphere Reserve located in Lam Dong Province, Vietnam, as a new species named Ophiocordyceps langbianensis,
belonging to the genus of Ophiocordyceps (Ophiocordycipitaceae, Hypocreales).
Table 3. DL0017 clustered together with Ophiocordyceps brunneipunctata with bootstrap support.
Gene Bootstrap value (NJ/MP/ML)
nrLSU
nrSSU
Rpb1
ITS
Tef
Figure4. Phylogenetic relationship between O. langbianensis and its allies based on ve regions, nrLSU-nrSSU-
Rpb-ITS-tef data. Bootstrap values (1,000 replicates) are indicated above the nodes.
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Received: 12 April 2020; Accepted: 18 November 2020
References
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Figure5. Reconstruction of Neighbor-Net network of DL0017 and its allies.
Table 4. Comparison between Ophiocordyceps langbianensis và Ophiocordyceps brunneipunctata. a Reference
from Ophiocordyceps brunneipunctata(Hywel-Jones) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora.
Ophiocordyceps langbianensis Ophiocordyceps brunneipunctataa
Stromata Arising from the head of host larva
Solitary, rarely branch, 40–100mm long Arising from one end of the insect larva
Solitary, rarely up to 3, simple, 25–90mm long
Stipe Fibrous, cylindrical 30–67mm × 0.7–1.0mm, light yellow Simple, cylindric, 5–15mm × 1–1.8mm, base reddish-brown
Fertile portion cylindrical 7.0–14.0mm × 1.5–2.0mm, brownish yellow with dark brown dots,
that present in the ostiole of the perithecia Subterminal, cinnamon in color, with brown ostioles apparent, 5–15 × 1–1.8mm
Perithecia Embedded, ovate or pyriform, 260–400μm × 100–190µm Immersed, ovate to pyriform, brown, 270–335μm × 110–160μm
Asci Cylindric, 200–250μm × 5.0–6.0μm, with thick cap Hyaline, cylindrical, 280–295μm × 6–7μm, with prominent apical cap
Ascospores Filiform, multiseptate, disarticulating into unicellular partspores
Partspores: cylindric, swollen, two waist-lilce constriction, 5.0–7.5μm × 1.25–
2.0µm
Hyaline, liform, exuous, breaking into partspores
Partspores truncate, 4–6μm × 1–1.5μm
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Acknowledgements
We express our special thanks to National Foundation for Science and Technology Development
(NAFOSTED)under the grant number106-NN.06.2015.44, Vietnam; Ho Chi Minh City Open University for
the genuine support throughout this research workunder the grant number E2019.06.3. We also thank Dr. Hiep
Minh Dinh, Son Kim Hoang, Hanh Van Trinh, Mai Hoang Nguyen, and Dr. Tien Van Tran for their assistance.
Author contributions
N.B.T. collected the sample DL0017. T.D.L., T.A.H.L. conceived, planned and carried out the experiments and
contributed to the interpretation of the results; T.D.L. took the lead in writing the manuscript. All authors pro-
vided critical feedback and revised the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Correspondence and requests for materials should be addressed to N.B.T.
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