Morphology and molecular study of three new Cordycipitoid fungi and its related species collected from Jilin Province, northeast China

Abstract Cordyceps species are notable medicinal fungi in China, which are pathogenic on insects and exhibit high biodiversity in tropical and subtropical regions. Recently, three new Cordyceps species, Cordycepschangchunensis and Cordycepsjingyuetanensis growing on pupae of Lepidoptera and Cordycepschangbaiensis growing on larvae of Lepidoptera, were found in Jilin Province, China and are described, based on morphological and ecological characteristics. These three new species are similar to the Cordycepsmilitaris group, but are distinctly distinguishable from the known species. Cordycepschangchunensis, characterised by its small and light yellow to orange stromata which is occasionally forked, covered with white mycelium at the base of stipe, globose to ovoid perithecia, is macroscopically similar to Cordycepsmilitaris. Cordycepschangbaiensis is clearly discriminated from other Cordyceps species by its white to orange and branched stromata, clavate to cylindrical fertile apical portion, immersed and globose to ovoid perithecia. Moreover, unbranched, clavate and orange to light red stromata, almond-shaped to ovoid and immersed perithecia separate Cordycepsjingyuetanensis from other Cordyceps species. nrITS, nrLSU and EF-1α sequences were undertaken and phylogenetic trees, based on Maximum Likelihood and Bayesian Inference analysis showed that the three new species clustered with Cordycepsmilitaris, but formed individual clades, as well as confirmed the results of our morphological study.


Introduction
The family Cordycipitaceae belongs to Hypocreales with plant-, animal-and fungusbased nutrition modes (Sung et al. 2007;Vega et al. 2009). The species of Cordycipitaceae are a wide variety which infect invertebrates and, in the tropics and subtropics, are known to have the highest species diversity (Kobayasi 1941(Kobayasi , 1982. According to current data, over 900 species of Cordycipitoid fungi are reported worldwide (Yan and Bau 2015;Zha et al. 2018). In China, more than 146 species are recorded (Yan and Bau 2015).
Cordycipitoid fungi were first described in 1753 as Clavaria militaris L., later being recognised as Cordyceps militaris (L.) Fr. The genus Cordyceps Fr. was established by Fries in 1818, encompassing over 450 species (Kobayasi 1982;Luangsa-ard et al. 2007). Compared with a large number of species, subdivisions into infrageneric groups, for example, subgenera and sections, have been proposed in the Cordyceps classification, traditionally based on morphological and ecological characters (Stensrud et al. 2005). The classification of Cordyceps, based on the studies of Kobayasi (1941Kobayasi ( , 1983, three subgenera, C. subg. Cordyceps, C. subg. Ophiocordyceps and C. subg. Neocordyceps were recognised. Subg. Cordyceps was characterised by the production of either immersed or superficial perithecia, which are approximately at right angles to the surface of stroma and ascospores break into part-spores at maturity. Mains proposed a different viewpoint, two subgenera, C. subg. Cryptocordyceps and C. subg. Racemella, were added (Mains 1958). Based on nrITS, nrSSU, nrLSU, EF-1α, RPB1, RPB2, TUB and ATP6 sequences, the phylogenetic study implied that the Cordycipitoid fungi belong to six genera (Cordyceps Fr., Metacordyceps G.H. Sung (Sung et al. 2007;Yan and Bau 2015).
The host of Cordycipitoid fungi is varied and the fungi are always parasitic on larvae of swifts, pupae of Lepidoptera, spiders etc. Cordycipitoid fungi have a strong relationship with the environment and its host (Zha et al. 2019).
In this study, three new species of Cordyceps are reported, based on morphology and molecular studies. Furthermore, the relationship between the host and Cordyceps species is analysed.

Sampling and morphological studies
The specimens were photographed in situ. The size of the stromata was measured when fresh. After examination and description of the fresh macroscopic characters, the specimens were dried in an electric drier at 40-45 °C.
Descriptions of macroscopic characters were based on field notes and photographs. The colours correspond to the "Flora of British fungi: colour identification chart" (Royal Botanic Garden 1969). The dried specimens were rehydrated in 94% ethanol for microscopic examination and then mounted in 3% potassium hydroxide (KOH), 1% Congo Red, Cotton Blue and Melzer's Reagent (Torres et al. 2005), along with a Zeiss Axio Lab. A1 microscope for observation. For each species, a minimum of 40 part-spores was measured from two different ascocarps, part-spores are given as length × width (l × w). The specimens examined are deposited in the Herbarium of Mycology of Jilin Agricultural University (HMJAU).

DNA extraction, PCR amplification and sequencing
Total DNA was extracted from dried specimens using the NuClean Plant Genomic DNA Kit (Kangwei Century Biotechnology Company Limited, Beijing, China). Sequences of the internal transcribed spacer region (ITS), nuclear large ribosomal subunits (LSU) and translation elongation factor 1-alpha (EF-1α) were used for phylogenetic analysis. The ITS sequence was amplified using the primer pair ITS4 and ITS5 (White et al. 1990), LSU sequence was amplified using the primer pair LROR and LR7 (Stensrud et al. 2005) and EF-1α sequence was amplified using the primer pair 983F and 2218R (Castlebury et al. 2004).
Reaction programmes followed Yan and Bau (2015), Castillo et al. (2018) and Ban et al. (2015), respectively. PCR products were visualised via UV light after electrophoresis on 1% agarose gels stained with ethidium bromide and purified using Genview High-Efficiency Agarose Gels DNA Purification Kit (Gen-View Scientific Inc., Galveston, TX, USA). The purified PCR products were sent to Sangon Biotech Limited Company (Shanghai, China) for sequencing using the Sanger method. The new sequences were deposited in GenBank.

Data analysis
Based on the results of BLAST and morphological similarities, the sequences obtained and related to these samples are listed in Table 1 (Thompson et al. 1997), MACSE V2.03 (Ranwez et al. 2018) and MAFFT (Katoh and Standley 2013), then manually adjusted in BioEdit (Hall 1999). The datasets were aligned first and then, nrITS, nrLSU and EF-1α sequences were combined with Mesquite. The tree construction procedure was performed in PAUP* ver-

NBRC106331
Elaphomyces sp. JN943320 JN941408 (Schoch et al. 2012) sion 4.0b10 (Swofford 2002) as described by Jiang et al. (Jiang et al. 2011). All characters were equally weighted and gaps were treated as missing data. MrModeltest 2.3 was used to determine the best fitting substitution model for each dataset for Bayesian Inference, which was calculated with MrBayes 3.2.6 with a general time-reversible DNA substitution model and a gamma distribution rate variation across sites (Ronquist and Huelsenbeck 2003). Four Markov chains were run for two runs from random starting trees for four million generations until the split deviation frequency value was < 0.01 and trees were sampled every 100 generations. raxmlGUI 2.0 (Edler et al. 2020) was used for Maximum Likelihood (ML) analysis with 1,000 bootstrap replicates using the GTRGAMMA algorithm to perform a tree inference and search for optimal topology (Vizzini et al. 2015).

Phylogenetic analysis
The phylogenetic tree, based on ITS from Bayesian analysis, included sequences from 46 fungal samples representing 43 taxa and the results are shown in Fig. 1. According to the phylogenetic tree, the three new species gather into one branch with C. militaris, C. roseostromata Kobayasi & Shimizu, C. taishanensis B. Liu, P.G. Yuan & J.Z. Cao, C. kyushuensis A. Kawam. and C. hepialidicola Kobayasi & Shimizu, but the species C. jingyuetanensis does not gather into one branch by itself. Meanwhile, the genus Cordyceps was divided into three independent clades. Furthermore, Cordyceps and Akanthomyces Lebert are a sister clade to Blackwellomyces Spatafora & Luangsa-ard.

Cordyceps militaris BCC55820/TBRC7276
Ophiocordyceps rubiginosiperit NBRC106966 For these reasons, the combined ITS, LSU and EF-1α dataset including 121 fungal samples representing 48 taxa was used for analysis and the results are shown in Fig. 2. In these data, the three new species are in three independent clades included in the C. militaris complex, C. jingyuetanensis was close to C. hepialidicola Kobayasi & Shimizu and is different from Fig. 1. From the phylogenetic tree (Fig. 2), the species of Cordyceps are mainly divided into three independent clades. Moreover, the family Cordycipitaceae clustered into three clades and the genus Akanthomyces formed a sister clade to the genus Cordyceps. Etymology. changchunensis: referring to Changchun, the location of the holotype. Diagnosis. Cordyceps changchunensis can be easily differentiated from closely-related species C. militaris by its unique host, smaller stromata, immersed perithecia and larger part-spores (2.6-6 × 1.0-1.4 μm).

Cordyceps changbaiensis
Description. Sexual Morph. Stromata 2.4-5.2 cm long, single or multiple, solitary, arising from the head of the host insect covered with white mycelia. Fertile apical portion, orange, clavate to cylindrical, 0.6-1.5 cm long and 0.2-0.6 cm wide, obviously distinguishable from the stipe. Sterile stipe fleshy, white to light yellow, cylindrical, 1.8-3.7 cm long and 0.  Ping Zhao (HMJAU 482260, isotype, GenBank Acc. nos.: ITS = MW893270, LSU = MW893272, EF-1α = MZ616774) Note. C. changbaiensis has orange to white and branched stromata. Morphologically, C. roseostromata Kobayasi & Shimizu is similar to C. changbaiensis due to the single or branched stromata. C. kyushuensis A. Kawam. is also close to C. changbaiensis because of the host and the stromata being similar in colour. However, both C. roseostromata and C. kyushuensis have a larger perithecia and smaller part-spores. Furthermore, the stromata of C. kyushuensis is gregarious or fascicled and grows from the head or abdomen of the host (Li et al. 2015); C. roseostromata has pyriform perithecia and host on larva of Coleoptera (Kobayasi 1983). In the phylogenetic analysis, C. changbaiensis was placed in separate monophyletic lineages (BPP = 0.95, MLBS = 97%) and formed a sister relationship with C. rosea. Etymology. jinyuetanensis: referring to Jingyuetan National Forest Park, the location of the holotype.
Diagnosis. C. jingyuetanensis is different from other species by growing on pupae, orange to light red stromata, immersed and almond-shaped to ovoid perithecia.
Description. Sexual Morph. Stromata 4-4.5 cm long, multiple, solitary, arising from pupae of Lepidoptera. Fertile apical portion, orange to light red, clavate, 0.8-1.3 cm long and 0.1-0.2 cm wide, obviously distinguishable from the stipe. Sterile stipe fleshy, light yellow to orange, cylindrical, 2.7-3.7 cm long and 0.1-0.2 cm wide, usually with white mycelium at the base. Perithecia immersed to the surface of the fruiting body, almond-shaped to ovoid, 220-340 × 110-220 μm, with a thick wall about 15-20 μm. Asci cylindrical, 225-475 × 3-5 μm, 8-spored, apex of ascus hemispherical to irregular, 3.0-4.0 × 1.4-2.8 μm. Part-spores oblong, 2.8-5.0 × 1.0-1.4 μm, smooth, hyaline in 3% KOH, thin-walled, inamyloid.   on the pupae of Diptera, I. cicadae grows on the pupae of Hemiptera and the stromata of C. qingchengensis are yellow, single or branched on the top. C. hepialidicola Kobayasi & Shimizu from Japan is similar to C. jingyuetanensis in its phylogenetic relationship, but there are distinct morphological differences. Morphologically, the stromata of C. hepialidicola are multiple, branched on the top sometimes, grow from the head of larva of Hepialida or Lepidoptera, have larger perithecia (300-350 × 500 μm) and smaller part-spores (3-4 × 1 μm) (Kobayasi 1983). In the phylogenetic analysis, C. changbaiensis was placed in separate monophyletic lineages (BPP = 0.92, MLBS = 79%). Note. C. militaris is a widely distributed species and also a well-known medicinal fungus in China. At this time, we collected samples from many different places. The morphological evidence shows no apparent differences between each other. However, the habitat is markedly different.   (Yan and Bau 2015). ML and BI analysis recognised four well-supported clades, one is Cordycipitaceae, the others are Clavicipitaceae and Ophiocordycipitaceae (Fig. 2). Moreover, the Cordycipitaceae branch is mainly divided into three clades, the Akanthomyces clade near the Cordyceps clade, implies a closer biological relationship. The previous studies have revealed that the genus Cordyceps was not monophylic (Artjariyasripong et al. 2001), the species of Isaria was nested within Cordyceps (Kepler et al. 2017) and our phylogenetic analysis also shows a similar result. Cordyceps clade consisted of three major subclades designated as clade 1, clade 2 and clade 3 (Fig. 2). Nearly all the subclades in Cordyceps clade were strongly supported.
Clade 1, including nine Cordyceps spp. and two Isaria spp. I. cicadae, based on Chinese sequences, gathers into one branch with Cordyceps species. What is known as I. cicadae in China, named on a Brazilian specimen, is of confused classification status, due to the teleomorph having remained undiscovered. In China, C. cicadae Massee has been regarded as a teleomorph of I. cicadae as well as a teleomorph of O. sobolifera (Hill ex Watson) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora and referred to as C. sobolifera (Hill ex Watson) Berk. & Broome. Until recently, the teleomorph was discovered in Mt. Jinggang, Jiangxi Province, China and both teleomorph and anamorph existed on some specimens, with the morphology of the anamorph consistent with those, "I. cicadae", harvested throughout southern China, significantly different from the type specimen of I. cicadae. For this reason, it was published as a new species named C. chanhua Z.Z. Li, F.G. Luan, Hywel-Jones, C.R. Li & S.L. Zhang (Zhi et al. 2021). Furthermore, I. japonica Yasuda reported from Japan, exhibits exceptionally high affinity with the genus Cordyceps. The teleomorph, however, still remains a mystery and a more intensive study is needed. Clade 2 consists of C. scarabaeicola Cordyceps spp. of clade 3A all arise from pupae. Clade 3B includes 11 Cordyceps spp., seven known Cordyceps spp., one unidentified Cordyceps sp. and our three new species. Being visually similar to Cordyceps militaris seems to be a synapomorphic character of clade 3B.
About 60% of Cordyceps sensu lato species are recorded on two insect orders-Coleoptera and Lepidoptera (Shrestha et al. 2016). Host preferences have been variously implemented in taxonomic work, so this is also in Cordyceps. Host associations, when superimposed on phylogeny, suggested that some groups of taxa have conserved the endoparasite-host interactions to some extent; however, several host shifts have occurred during the evolution of Cordyceps (Stensrud et al. 2005). In Cordyceps species, hosts were considered as having low significance as a phylogenetic character, but are the most crucial feature in morphological aspects (Torres et al. 2005).