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

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.


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 . The macroscopic characteristics of the fresh fruit body were carefully observed, including the stipe, stroma, etc. Moreover, the color was noted according to Kornerup and Wanscher 8 . Additionally, the host insect was identified 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 amplification, target gene sequencing. Genomic DNA was isolated by using the phenol/chloroform method (pH = 8) 11 . The fruiting body was incubated in a lysis buffer (2.0% SDS, Tris-HCl pH 8.0, 150 mM NaCl, 10 mM EDTA, 0.1 mg/ml Proteinase K) at 65 °C overnight. The supernatant was collected by centrifugation, and a volume of 700 μL of phenol/chloroform/isoamyl alcohol (25:24:1) was supplemented and centrifuged. The supernatant was collected and precipitated with absolute isopropanol. Finally, the isolated genomic DNA was stored in Tris-EDTA buffer at − 20 °C for further studies.
The primer pairs used to amplify nrLSU, nrSSU, rpb1, ITS and Tef regions are shown in Table 1. The final 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 temperatures for each target gene shown in Table 1); 5 μL aliquots of amplification product were electrophoresed on a 2.0% agarose gel and visualized in a UV transilluminator. The amplified product was sequenced at Nam Khoa (Vietnam) company.
Taxa and nrLSU, nrSSU, rpb1, ITS and tef sequences collection, DNA proofreading and phylogeny analysis. The 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 . The nrLSU, nrSSU, rpb1, ITS and tef were noted with accession number, name of taxon and locality. The amplified DNA sequences were proofread to remove ambiguous signals at both ends by different software, including Seaview 4.2.12 and Chromas Lite 2.1.1. The phylogenetic tree was constructed based on neighbor-joining (NJ), maximum parsimony (MP), and maximum likelihood (ML), using Molecular Evolutionary Genetics Analysis (MEGA) version 5. Additionally, the best evolution model was predicted using jModelTest.
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.
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 study 7 . The 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 3). Information from molecular phylogenetic analysis based on separate genes is not enough to reconstruct trees for higher classification compared to multigene analysis. Therefore, a combined data set, including 2,319 bp of five target genes, nrLSU-nrSSU-Rpb1-ITS-tef, was analyzed. The evolution model that was most fixed with the combined dataset was TN93 + G + I, as determined by MEGA 5.2. The 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). The molecular phylogenetic analysis confirmed that there were differences between DL0017 and other related species.
To confirm 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. The Neighbor-Network analysis supported the results from the phylogenetic analysis (Fig. 5). The network presented three complex groups, corresponding to three families: Clavicipitaceae, Ophiocordycipitaceae and Cordycipitaceae. The 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, suggesting a close relationship. To confirm the authenticity of DL0017 as a new species, we compared DL0017 and its close species, O. brunneipunctata. It differed from O. brunneipunctata by the morphological characteristics described in Table 4. Therefore, DL0017 was confirmed as a new species, namely O. langbianensis.

Discussion
Lang Biang Biosphere Reserve, located in Lam Dong Province, is classified 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, fibrous, pliant, and rarely fleshy. Additionally, asci are usually cylindrical with thickened ascus apex. Ascospores are usually cylindrical, multiseptate, and disarticulate into part-spores or non-disarticulating 7 . Our specimen shares these common characteristics.
Based on the phylogenetic analysis, the specimen DL0017 clustered with Ophiocordyceps brunneipunctata in Ophiocordycipitaceae 12 . However, the morphologies of these two species are different in many characteristics, including color, size of stroma, stipe, and dots in the fertile portion. The 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. langbianensis. The stroma of Cordyceps furcicaodata arose from the middle of the host larva, while that of O. langbianensis arose from one end of the insect larva 13 . 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 often break into unicellular part-spores. www.nature.com/scientificreports/ www.nature.com/scientificreports/ The asexual morph of O. langbianensis consists of long and divergent phialides, elliptical conidia usually in chains considered paecilomyces-like or purpureocillium-like 14,15 . Conversely, O. bruneipunctata produced a mononematous hirsutella-like asexual morph from colonies after 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 Biosphere Reserve located in Lam Dong Province, Vietnam, as a new species named Ophiocordyceps langbianensis, belonging to the genus of Ophiocordyceps (Ophiocordycipitaceae, Hypocreales).  www.nature.com/scientificreports/