Ophiocordycepsaphrophoridarum sp. nov., a new entomopathogenic species from Guizhou, China

Abstract Background Ophiocordyceps is the largest genus in the family Ophiocordicipitaceae, including many entomopathogenic species. In recent years, many species have been described in this genus, with a wide range of host insects. Entomopathogenic fungi include ecologically, economically and medicinally important species, but a large portion of their diversity remains to be discovered and described. New information In this study, a new species, Ophiocordycepsaphrophoridarum sp. nov, parasitising Aphrophoridae sp. (Hemiptera) is proposed from China, based on evidence from morphology and molecular phylogenetic analyses. This species is characterised by fibrous, pigmented stromata, cylindrical asci and filiform ascospores. Compared to its closest relative, O.tricentri, the new species has wider perithecia and longer asci. Molecular phylogenetic analyses of a multilocus dataset (consisting of SSU, ITS, LSU, TEF1, RPB1 and RPB2) confirm its placement in Ophiocordyceps. Ophiocordycepsaphrophoridarum is morphologically described and illustrated with colour photographs. Morphological comparisons with closely-related species are also presented in tabulated format.


Introduction
Insect-associated fungi represent a largely unknown and undescribed group; only 1.5% of these fungi have been reported (Mueller and Schmit 2007). In 2019, scientists determined 48 new species of animal-associated Sordariomycetes, including eight species of Ophiocordyceps, one of the best-known entomopathogenic genera (Cheek et al. 2020).
The genus Ophiocordyceps was proposed by Petch 1931) and was originally considered as a subgenus of Cordyceps (Kobayasi 1941, Kobayasi andShimizu 1983). Sung et al. (2007b) established Ophiocordycipitaceae as a new family in Hypocreales with Ophiocordyceps as type genus. Due to the polyphyletic nature of Cordyceps, species formerly assigned to this genus had to be recombined in Ophiocordyceps ( Sung et al. 2007a, Johnson et al. 2009). To date, Ophiocordyceps is the most speciose genus in Ophiocordycipitaceae with 289 accepted species (Index Fungorum, accessed 11 March 2021). Species of Ophiocordyceps are characterised by producing fibrous, hard, flexible, pigmented stromata and cylindrical asci with apical caps (Sung et al. 2007a, Ban et al. 2015, Maharachchikumbura et al. 2015, Wijayawardene et al. 2017, Xiao et al. 2019. The asexual morph of Ophiocordyceps is linked to Hirsutella, Hymenostilbe, Paraisaria, Stibella and Syngliocladium ( Sung et al. 2007a, Thanakitpipattana et al. 2020) and known as Hirsutella-like and Hymenostilbe-like (Kepler et al. 2013, Maharachchikumbura et al. 2016, Maharachchikumbura et al. 2015. Species in Ophiocordycipitaceae are found on a wide range of insect hosts; some taxa are host specific, such as Ophiocordyceps unilateralis sensu lato ( De Bekker et al. 2014, Kobmoo et al. 2019. Blattaria, Coleoptera, Dermaptera, Diptera, Hemiptera, Hymenoptera, Isoptera, Lepidoptera, Megaloptera, Neuroptera, Odonata and Orthoptera are the insect orders most commonly reported to be associated with Ophiocordyceps (Evans et al. 2011, Araujo and Hughes 2019. The functional morphology of Ophiocordyceps is diverse and considered to be exclusively related to the host's ecology and biology (Evans et al. 2011).
Ophiocordyceps has a pan-global distribution, but is most species-rich in the tropics and subtropics (Petch 1933, Petch 1937, Kobayasi 1941, Tzean et al. 1997, Ban et al. 2015. The Yuntai Mountain Nature Reserve, China, a dolomite karst landform, has become a hotspot for fungal diversity (Luo et al. 2013, Wen et al. 2015) and, in 2019, samples of Ophiocordyceps were collected that proved to be an undescribed species. Here, we formally describe this species, based on morphological study and the phylogenetic analysis of a multilocus dataset.

Collection and morphological characteristics examination
Two fresh samples of Ophiocordyceps, parasitising Aphrophoridae sp. (Hemiptera), were collected in June 2019 from the broad-leaved forest in Yuntai Mountain Nature Reserve, Guizhou Province, China. The samples were dried with silica gel and then stored in plastic boxes in the Herbarium of Mae Fah Luang University (MFLU). For micro-morphological observations, ascomata were examined using a Motic SMZ 168 Series stereomicroscope (Motic, Xiamen, China). Structures were observed and measured after being sliced with a double-sided blade and placed into water. Microphotographs were taken using an Eclipe 80i compound microscope (Nikon, Tokyo, Japan), fitted with an EOS 600D camera (Canon, Tokyo, Japan). Measurements were made using the Tarosoft (R) Image Frame software v. 0.9.7.

DNA extraction, PCR amplification and determination of DNA sequences
DNA was extracted from dried fruiting bodies using the Fungal gDNA Kit (Biomiga, Sang Diego, CA, USA). We amplified the small and large subunits (SSU, LSU) of the ribosomal RNA gene, internal transcribed spacer region (ITS), translation elongation factor-1α (TEF1) and the largest and second-largest subunit of RNA polymerase II gene (RPB1, RPB2). The following primer pairs were used: NS1/NS4 for SSU, ITS4/ITS5 for ITS, LR0R/LR5 for LSU (Hopple and Vilgalys 1994, Vilgalys and Hester 1990, White et al. 1990), EF1-983F/ EF1-2218R for TEF1 (Sung et al. 2007b), CRPB1A/RPB1Cr for RPB1 and fRPB2-6f/ RPB2-7CR for RPB2 (Castlebury et al. 2004). The 25-μl PCR reaction volume contained 2 μl of DNA template, 8.5 μl of H O, 1 μl of each forward reverse primer and 12.5 μl of 2× benchtoptm Taq Master Mix (Biomiga, San Diego, CA, USA). Cycling conditions were as follows: for SSU and LSU: initial denaturation at 94°C for 3 min; followed by 33 cycles at 94°C for 30 s, 51°C for 30 s and 72°C for 2 min; and final extension at 72°C for 10 min. For ITS: initial denaturation at 94°C for 3 min; followed by 33 cycles of 94°C for 30 s, 51°C for 50 s and 72°C for 45 s; and final extension at 72°C for 10 min. For TEF1: initial denaturation at 94°C for 3 min; followed by 33 cycles of 94°C for 30 s, 58°C for 50 s and 72°C for 1 min; and final extension at 72°C for 10 min. For RPB1: initial denaturation at 94°C for 3 min; followed by 33 cycles of 94°C for 1 min, 52°C for 1 min and 72°C for 1 min; and final extension at 72°C for 10 min. Lastly, for RPB2: initial denaturation at 94°C for 3 min; followed by 33 cycles of 94°C for 30 s, 54°C for 40 s and 72°C for 80 s; and final extension at 72°C for 10 min. Amplified PCR products were verified by 1% agarose gel electrophoresis, stained with ethidium bromide in 1× TBE. The PCR products were sequenced by Shanghai Shenggong Biological Engineering Co. (Hangzhou, Shanghai, China). Forward and reverse sequence reads were assembled and edited by BioEdit v. 7.0.9 (Hall et al. 2011).
Maximum Likelihood (ML) analyses were performed using IQ-TREE 2 (Minh et al. 2020) under partitioned models; the built-in ModelFinder (Kalyaanamoorthy et al. 2017) was used to select appropriate models for each of the six loci. Branch support was estimated using 1000 ultrafast bootstrap (UFBoot2) replicates (Hoang et al. 2018). Bayesian Inference (BI) was determined by Markov Chain Monte Carlo (MCMC) sampling using MrBayes v.3.1.2 (Ronquist et al. 2012). The six loci were concatenated into a single dataset. BI was performed with six independent MCMC runs and trees were sampled every 100 generation. The analyses were stopped after 5,000,000 generations when the average standard deviation of split frequencies was below 0.01. The convergence of the runs was checked using Tracer v.1.6 (Rambaut et al. 2018). The first 25% of the resulting trees were discarded as burn-in and posterior probabilities (PP) were calculated from the remaining sampled trees. The ML tree was visualised with FigTree v.1.4.0 (http://tree.bio.ed.ac.uk/ software/figtree/).

Etymology
Referring to the host, Aphrophoridae sp.

Distribution
Thus far only known from China.

Host
Aphrophoridae sp. (Hemiptera), collected from the underside of leaves litter, stromata growing from the prothorax.

Analysis Phylogenetic analyses
A total of 185 sequences, representing 128 species of Ophiocordycipitaceae, were downloaded from GenBank. The final alignment length was 4412 characters, representing 185 taxa (822 for LSU, 481 for ITS, 919 for SSU, 918 for TEF1, 536 for RPB1 and 736 for RPB2) (Suppl. materials 1, 2). Tree topology of the IQ-TREE analysis was similar to the one from the Bayesian analyses. The best-scoring ML (-lnL = 81595.8951) is shown in Fig. 2.

Discussion
The Yuntai Mountain Nature Reserve, situated in Shibing County, Guizhou Province, China, is a dolomite karst landform. The Reserve is home to 106 species of macrofungi (Luo et al. 2013), including two species of Metacordyceps that are currently only known from the holotype locality (Wen et al. 2015 Here, we present a new entomopathogenic species, O. aphrophoridarum, from the same Reserve.    -Jones 1995). This species produces larger ascomata, longer asci and longer partspores compared to O. aphrophoridarum (Shrestha and Sung 2005). Additionally, O. vespulae has Vespula sp. as host (Hymenoptera) and is distinct from the new species by its longer asci and partspores (Long et al. 2021).
Ophiocordyceps tricentri is phylogenetically most closely related to the new species and it has similar morphological characters. Ophiocordyceps tricentri was initially described as Cordyceps tricentri from Japan. It is characterised by stipitate stroma with a yellow fusoid fertile head (Yasuda 1922, Table 1). The host of C. tricentri was initially identified as Tricentrus sp. (Hemiptera, Membracidae), but later corrected to Aphrophora intermedia (Hemiptera, Aphrophoridae) (Yasuda 1922). Later, Aphrophora flavomaculata, Aphrophora rugosa and Peuceptyelus medius were reported as the hosts of C. tricentri ( Kobayasi 1941, Shrestha 2017. Additionally, another species, Cordyceps aphrophorae, was synonymised with C. tricentri (Yasuda 1922, Lim andKim 1973). Shrestha and Sung 2005) recorded Cordyceps tricentri obtained from Nepal, but presented no molecular data (Table  1). Following molecular phylogenetic analyses, C. tricentri was transferred to Ophiocordyceps ( Sung et al. 2007b). Ban et al. 2015) presented sequence data of O. tricentri from strain NBRC 106968, but did not provide morphological information. It is clear that more data are needed to fully understand the species limits with regards to O. tricentri.
The new species, O. aphrophoridarum, is morphologically similar to O. tricentri, but can be recognised by its longer and finer stromata and much longer asci (Yasuda 1922, Shrestha andSung 2005, Table 1.

References
This study Yasuda 1922 Shrestha andSung 2005 In conclusion, there is sufficient evidence from both morphology and molecular phylogenetic analyses to support O. aphrophoridarum as a new species of Ophiocordyceps.