﻿Multigene phylogeny and morphology reveal Ophiocordycepshydrangea sp. nov. and Ophiocordycepsbidoupensis sp. nov. (Ophiocordycipitaceae)

﻿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 Ophiocordycepssobolifera 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. Ophiocordycepshydrangea and Ophiocordycepsbidoupensis were introduced based on morphology and multigene phylogenetic evidence. The phylogenetic framework of Ophiocordyceps was reconstructed using a multigene (nrSSU, nr LSU, tef-1α, rpb1, and rpb2) dataset. The phylogenetic analyses results showed that O.hydrangea and O.bidoupensis were statistically well-supported in the O.sobolifera clade, forming two separate subclades from other species of Ophiocordyceps. The distinctiveness of these two new species was strongly supported by both molecular phylogeny and morphology.


Introduction
Ophiocordyceps G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora is the largest genus in the Ophiocordycipitaceae, comprising approximately 290 species. It was originally established by Petch, with Ophiocordyceps blattae Petch as the type species (Petch 1931). According to the arrangement of the perithecia, the size of asci, ascospores, and secondary ascospores, Ophiocordyceps was transferred to Cordyceps sensu lato by Kobayasi, as a subgenus of Cordyceps s.l. (Kobayasi 1941(Kobayasi , 1982. Sung et al. (2007) used five to seven loci combined molecular datasets to revise the Cordyceps and the Clavicipitaceae. The species of Cordyceps and Clavicipitaceae were divided into three families (Cordycipitaceae, Ophiocordycipitaceae, Clavicipitaceae sense stricto) and four genera (Cordyceps sense stricto, Ophiocordyceps, Elaphocordyceps, and Metacordyceps). The research results of Sung et al. (2007) (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. 2021aWang et al. , 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. 2007Sung et al. , 2011Luangsa-ard et al. 2008;Hyde et al. 2017;Crous et al. 2018Crous et al. , 2019Lao et al. 2021;Wang et al. 2021a). Asexual morphs of Ophiocordyceps were reported as Hirsutella Pat., Paraisaria Samson & B.L. Brady, Sorosporella Sorokin, Hymenostilbe Petch and Syngliocladium Petch, etc. (Sung et al. 2007;Quandt et al. 2014). In most species of Ophiocordyceps, their dominant asexual morphs were Hirsutella, the conidiogenous cells basally swollen that taper to a narrow neck, producing a mucilaginous cluster of one or several conidia (Simmons et al. 2015;Wang et al. 2018).
Ophiocordyceps species have a wide range of insect hosts, from solitary beetle larvae to social insects. More than 10 insect orders were attacked, including Hemiptera, Coleoptera, Lepidoptera, Blattaria, Dermaptera, Diptera, Hymenoptera, Isoptera, Megaloptera, and Mantodea (Araújo et al. 2015;Hughes 2016, 2019). Entomopathogenic fungi whose hosts are cicada nymphs have attractive stromata. The most typical representative of this group was Cordyceps cicadae (Miquel) Massee (Massee 1895) in Cordycipitaceae, with the stroma like a flower (Sung et al. 2007). However, for species of Ophiocordyceps, with cicada nymph hosts including O. khonkaenensis Tasanathai (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. 2007Sung et al. , 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 (Chen et al. 2021). The unique geographical conditions of Yunnan have resulted in high Cordyceps s.l. species diversity. There is also a high species diversity of Cordyceps s.l. in Southeast Asia, where more than 500 species of entomopathogenic fungi have been reported. Approximately 400 species of entomopathogenic fungi are distributed in Thailand (Sung et al. 2007;Luangsa-ard et al. 2011Luangsa-ard et al. , 2018Ban 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 (Xu et al. 2022). These findings suggested that Vietnam should be abundant in species diversity of Cordyceps s.l. (Mongkolsamrit et al. 2011;Doan et al. 2017;Luyen et al. 2017).
Several studies have evaluated the taxonomy and biology of entomopathogenic fungi, especially species found in China and Southeast Asia. In this study, one unknown species of Ophiocordyeps attacking a cicada nymph was collected from Yunnan Province, Jinghong City, Nabanhe National Nature Reserve, in China. Another unknown species of Ophiocordyeps attacking larvae of Elateridae was collected from Lintong Province, Bidoup Nuiba National Park, in Vietnam. The phylogeny and morphology of these two fungi were determined, and their systematic position was established in Ophiocordycipitaceae. The phylogenetic analyses results showed that the two new species belonged to Ophiocordyceps, and were named Ophiocordyceps hydrangea and Ophiocordyceps bidoupensis based on well-supported morphology and molecular data.

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

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

Phylogenetic analyses
Sequences of the five genes (nrSSU, nrLSU, tef-1α, rpb1, and rpb2) were downloaded from GenBank, and combined with the newly generated sequences in this study. The taxa information of the species and GenBank accession numbers of the five genes are listed in Table1. Sequences of the five genes were aligned using the Clustal X (v.2.0) and MEGA6 (v.6.0) (Larkin et al. 2007;Tamura et al. 2013). Ambiguously aligned sites were eliminated, and the gaps were treated as missing data. The aligned sequences of the five genes (nrSSU, nrLSU, tef-1α, rpb1, and rpb2) were concatenated into a single combined dataset using MEGA6 (v.6.0.). Conflicts between the five genes were tested using PAUP* (v.4.0b10) (Swofford 2002). The results of the phylogenetic signals in the five genes were not in conflict. The concatenated dataset containing all five genes consisted of 11 data partitions, including one each for nrSSU and nrLSU, and three for each of the three codon positions of tef-1α, rpb1, and rpb2. Phylogenetic analyses based on the five genes were made using BI and ML methods (Ronquist and Huelsenbeck 2003;Stamatakis et al. 2008). We used the optimal model GTR+I with 1,000 rapid bootstrap replicates on the five genes for ML analyses (Stamatakis 2006

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,  Sexual morph. The stroma was grown from the head of the host cicada nymph, solitary, the top of the stroma similar to hydrangea, pale pink, 1.6-6.4 cm long. Sexual morph was not observed.
Asexual morph. The colony grew slowly on PDA medium. Cultured at 25 °C for about 12 weeks, the diameter of the colony was 25-28 mm, pale pink, the edge white, hard texture. The back of the colony was white to brown. Surface hyphae rough, hyaline, septate. Conidiophores were cylindrical. Conidiogenous cells were solitary or whorled, ampuliform, smooth-walled, forming on conidiophores or colonies, hyaline, with swollen base, and slender top, 10.6-17.6 µm long, 2.9-4.3 µm wide at the swollen base, and 1.1-2.2 µm wide at the slender top. Conidia hyaline, ovoid or long oval, solitary, 6.8-10.1 × 3.3-4.5 µm.
Habitat. The hosts were buried in soil, and the stroma were found in the leaf litter on the forest floor.
Distribution. Vietnam.   (Fig. 1). Their hosts were larvae of Elateridae compared to cicada nymph hosts of the other species of the O. sobolifera clade (Table 2). Ophiocordyceos bidoupensis was well-supported by bootstrap support and posterior probabilities, and formed a separate subclade with O. houaynhangensis, O. brunneipunctata, O. langbianensis, and O. cossidarum. The morphology of O. bidoupensis was clearly different in shape and size from other species of O. sobolifera clade ( Table 2). The stroma of O. bidoupensis grew solitary from the head of the host; sterile apices of the stroma were different from the other species.

Discussion
Ophiocordyceps is the largest genus in the Ophiocordycipitaceae, with a wide range of hosts and various species. At present, more than 290 species of Ophiocordyceps have been reported (Index Fungorum 2022). However, only 11 species are described in the O. sobolifera clade and their hosts are mainly Coleoptera larvae and cicada nymphs (Hemiptera) ( Table 2). We describe the new species O. hydrangea attacking cicada nymphs and the new species O. bidoupensis attacking Coleoptera larvae. Most species have diverse macro-morphological or micro-morphological characteristics due to the same entomopathogenic fungi having a different host, or different species of entomopathogenic fungi having the same host (Sung et al. 2007(Sung et al. , 2011Araújo et al. 2015;Araújo and Hughes 2016;Shrestha et al. 2016;Luangsa-ard et al. 2018;Crous et al. 2019;Fan et al. 2021;Wang et al. 2021a). Hemiptera hosts are widely present among the species of Ophiocordyceps, including species of the Hirsutella clade, O. sobolifera clade, O. sphecocephala clade, and O. ravenelii clade.
The entomopathogenic fungi whose host is Hemiptera have diverse morphological characteristics. For example, O. nutans (Patouillard) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora (Sung et al. 2007), its hosts were stink bugs (Hemiptera), stromata solitary or multiple, fertile parts was red (Hywel-Jones 1995a;Luangsa-ard et al. 2008), stromata of O. brunneinigra (Hemipteran host) were flexuous, arising from between the head and the thorax of the host , stromata of O. spataforae Tasanathai, Thanakipipattana, Khonsanit & Luangsa-ard were cylindrical, cream to pale brown . However, from previously reported Hemipteran hosts, only a few hosts of the O. sobolifera clade were cicada nymphs in Ophiocordyceps (Kobayasi and Shimizu 1963;Sung et al. 2011;Crous et al. 2019). In this study, the host of O. hydrangea was a cicada nymph. More interestingly, the O. hydrangea was significantly more beautiful than other species; the stroma grew from the head of the host cicada nymph, and the top of the stroma like a hydrangea (Sung et al 2007(Sung et al , 2011Crous 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 & (Sung et al. 2007). Most species with Coleopteran host occur in soil and have solid, cylindrical, and yellow stromata. This is consistent with the results of this study. Phylogenetic analyses based on the data from five genes showed that our phylogenetic framework of Ophiocordyceps was consistent with previous studies (Sung et al. 2007(Sung et al. , 2011Quandt et al. 2014;Simmons et al. 2015;Crous et al. 2018Crous et al. , 2019Wang et al. 2018Wang et al. , 2021aLao et al. 2021 and O. furcatosubulata in the same clade. Species within the O. sobolifera clade had different hosts, and morphological characteristics. These two new species clustered in two separate subclades within the O. sobolifera clade. The hosts of one subclade were cicada nymphs with stromata cylindrical or sarciniform, bright-colored, conidia were macro (Kobayasi and Shimizu 1963;Crous et al. 2019), and the hosts of another subclade were Coleoptera, with stromata cylindrical, conidia small, and a sterile apex on top of the stroma (Hywel-Jones 1995b; Luangsa-ard et al. 2008;Crous et al. 2018;Lao et al. 2021;Wang et al. 2021a). Therefore, the species of the O. sobolifera clade could be divided into two separate subclades when more materials were collected.
The species of O. sobolifera clade had diverse morphological characteristics ( Table 2). The entomopathogenic fungi with cicada nymph hosts shared similar characteristics, stromata solitary or multiple, cylindrical, and bright-colored. However, they also differed in morphology. For example, O. sobolifera lacked a protruding ostiole with immersed perithecia (Kobayasi and Shimizu 1963), and this seems to be contrary to O. yakusimensis (Kobayasi and Shimizu 1963). Stromata of O. longissima were longer than other species, and had a short neck in perithecia (Sung et al. 2011). Compared to the ovoid perithecia of O. longissima and O. yakusimensis, O. khonkaenensis was flask-shaped (Crous et al. 2019). The top of the stroma of O. hydrangea was similar to hydrangea, the size and shape of conidiogenous cells and conidia were different from O. khonkaenensis (Table 2). The entomopathogenic fungi using Coleoptera hosts shared similar characteristics, such as stromata solitary, cylindrical, sterile apices on top, bright-colored. However, they had different shape and size of perithecia, asci, ascospores, conidiogenous cells, and conidia. The perithecia of O. bidoupensis was pyriform to lanceolate and brown-yellow. It was similar to O. brunneipunctata, O. furcatosubulata, and O. langbianensis, and only O. houaynhangensis was clavate (Hywel-Jones 1995b;Luangsa-ard et al. 2008;Crous et al. 2018;Lao et al. 2021;Wang et al. 2021a). Conidiogenous cells of O. bidoupensis were cone-shaped, forming on hyphae, with a hypertrophic base, tapering abruptly into a thin neck, smoothwalled, with a smaller thin neck (0.42 µm wide) than O. brunneipunctata (0.5 µm), O. furcatosubulata (0.9 µm), and O. houaynhangensis (0.5 µm).
Due to the unique geographical locations and climate conditions in China and Vietnam, these areas contain a rich species diversity of Cordyceps s.l. However, our survey of Cordyceps s.l. in China and Vietnam only represented a small portion of the total. More samples of Cordyceps s.l. will continue to be collected in China and Southeast Asia in order to uncover additional undescribed taxa, and revise species with the incorrect classification position of this group.