683 MycoKeys MycoKeys 121: 253-270 (2025) DOI: 10.3897/mycokeys.121.156843 Research Article Two new species and one asexual morph record of Paraisaria (Ophiocordycipitaceae, Hypocreales) from China Yu Yang'224©, Kevin D. Hyde?5°®, Ausana Mapook®®, Yong-Zhong Lu'”®, Somrudee Nilthong™®, Shu-Qiong Xie'”®, Xiang-Dong Li'™®, Ruvishika S. Jayawardena**’”®, Yuan-Pin Xiao'?© NN Do FP WwW YH School of Food and Pharmaceutical Engineering, Guizhou Institute of Technology, Guiyang 550025, China Guizhou Key Laboratory of Agricultural Microbiology, Guizhou Academy of Agricultural Sciences, Guiyang 550009, China Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand School of Science, Mae Fah Luang University, Chiang Rai, 57100, Thailand Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 22452, Riyadh 11495, Saudi Arabia Department of Plant Pathology, College of Agriculture, Guizhou University, Guiyang, Guizhou 550025, China Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul, 02447, South Korea Corresponding authors: Ruvishika S. Jayawardena (ruvishika.jay@mfu.ac.th); Yuan-Pin Xiao (emmaypx@gmail.com) OPEN Qaceess Academic editor: Ning Jiang Received: 24 April 2025 Accepted: 24 July 2025 Published: 1 September 2025 Citation: Yang Y, Hyde KD, Mapook A, Lu Y-Z, Nilthong S, Xie S-Q, Li X-D, Jayawardena RS, Xiao Y-P (2025) Two new species and one asexual morph record of Paraisaria (Ophiocordycipitaceae, Hypocreales) from China. MycoKeys 121: 203-270. https://doi.org/10.3897/ mycokeys.121.156843 Copyright: © Yu Yang et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Abstract Paraisaria is a genus within Ophiocordycipitaceae, primarily parasitising insect groups such as ants (Hymenoptera), moth larvae (Lepidoptera) and beetle larvae (Coleoptera). The genus is characterised by cylindrical stipes, subglobose to globose fertile heads with immersed perithecia, hyaline, multi-septate ascospores and irregularly branched conidio- phores with flask-shaped phialides and cylindrical to fusiform conidia. Paraisaria is glob- ally distributed, primarily inhabiting tropical and subtropical locations; however, it has also demonstrated adaptability to temperate climates. This study introduces two novel species and reports one asexual morph of Paraisaria from China, providing detailed descriptions, illustrations and molecular phylogenetic analyses. Morphological examination reveals clear distinctions between the new species and previously described taxa. Multi-locus phylogenetic analyses (LSU, ITS, SSU, tef-1a, rpb1 and rpb2) corroborate their uniqueness, offering new insights into the diversity and evolutionary dynamics of the genus. Key words: Entomopathogenic fungi, morphology, multi-locus, phylogeny, taxonomy Introduction Paraisaria, a genus within Ophiocordycipitaceae (Hypocreales, Hypocreomyceti- dae, Sordariomycetes, Pezizomycotina, Ascomycota, Fungi, Hyde et al. (2024)), was established by Samson and Brady with P. dubia as the type species (Samson and Brady 1983). Its sexual morph was previously recognised as Ophiocordyceps gracilis (syn. Cordyceps gracilis) (Samson and Brady 1983). Early studies linked its asexual morphs to insect larvae and successfully isolated them from the sex- ual morphs of Ophiocordyceps (Samson and Brady 1983; Li et al. 2004; Sung et al. 2007). Initially, Paraisaria was proposed for suppression, favouring a broader con- cept of Ophiocordyceps under the “one fungus, one name” principle to unify sexu- al and asexual classifications (Quandt et al. 2014). However, molecular analyses 253 Yu Yang et al.: Three new species of Paraisaria (Ophiocordycipitaceae, Hypocreales) from China revealed Paraisaria as a distinct monophyletic clade within the “Ophiocordyceps ravenelii subclade” (Sanjuan et al. 2015). Paraisaria was ultimately resurrected, segregated from Ophiocordyceps and amended to include sexual morphology, as proposed by Mongkolsamrit et al. (2019). Following this re-instatement, the genus has been further supported by the description of new species and combi- nations in subsequent studies (Wei et al. 2021; Tehan et al. 2023). Paraisaria parasitises insect hosts, including cicada nymphs (Hemiptera), larvae of beetles (Coleoptera), flies (Diptera), moths (Lepidoptera) and ants (Hymenoptera), with habitats ranging from soil to leaf litter (Kobayasi 1941; Ev- ans et al. 2010; Mongkolsamrit et al. 2019; Tehan et al. 2023). Geographically, Paraisaria has a broad distribution, predominantly in tropical and subtropical re- gions, such as Brazil, China and Argentina, but also occurs in temperate zones including parts of Europe and North America (Kobayasi 1941; Evans et al. 2010; Wen et al. 2016; Mongkolsamrit et al. 2019; Wei et al. 2021; Tehan et al. 2023). This wide range suggests adaptability to various ecological conditions, with a preference for warm and humid environments (Hennings 1904; Kobayasi 1941; Mongkolsamrit et al. 2019). In addition to its ecological versatility, Paraisaria is defined by unique mor- phological features in both sexual and asexual forms. Sexual morphs are char- acterised by cylindrical, fleshy stipes, subglobose to globose fertile heads with immersed perithecia, cylindrical asci with thickened apical caps and hyaline, multi-septate ascospores fragmenting into cylindrical part-spores (Samson and Brady 1983; Mongkolsamrit et al. 2019; Tehan et al. 2023). Asexual morphs, documented in eight species, feature irregularly branched conidiophores with flask-shaped phialides and cylindrical, ellipsoid or fusiform conidia (Mongkol- samrit et al. 2019; Wei et al. 2021). Paraisaria includes fungi that are important for ecology and the economy, yet their taxonomic diversity and ecological roles are still inadequately investigated (Mongkolsamrit et al. 2019; Tehan et al. 2023). Amongst the few known species, Paraisaria gracilis is particularly notable for its traditional use in Kazakh medi- cine and its anti-oxidative and antibacterial properties (Ma et al. 2012; Huang et al. 2019; Suo et al. 2014). Conversely, some species, such as P. heteropoda, pose public health risks due to their parasitism of edible insects, including cica- das (Doan et al. 2017). These fungi have been implicated in food poisoning out- breaks, including fatalities, caused by toxic mycotoxins, such as ibotenic acid (Doan et al. 2017; Tehan et al. 2023). Recent integrative analyses combining DNA sequencing with LC-HRMS techniques have further unveiled the genus’s chemical diversity (Tehan et al. 2023). Despite these advances, the taxonomic boundaries and species diversity within Paraisaria remain poorly resolved, hin- dering a comprehensive understanding of its ecological and economic poten- tial (Doan et al. 2017; Mongkolsamrit et al. 2019; Tehan et al. 2023). This study expands the understanding of Paraisaria by discovering two novel species and reports one asexual morph record from China. Morphological ex- aminations revealed distinct traits that differentiate these species from known members of the genus. Phylogenetic analyses, based on six loci (LSU, ITS, SSU, tef-1a, rpb1 and rpb2), confirmed their novelty and classification within Parais- aria. These findings contribute to the growing knowledge of fungal diversity and highlight the evolutionary relationships within the genus, while also emphasising the need for further research on its ecological roles and life cycle mechanisms. MycoKeys 121: 253-270 (2025), DOI: 10.3897/mycokeys.121.156843 954 Yu Yang et al.: Three new species of Paraisaria (Ophiocordycipitaceae, Hypocreales) from China Materials and methods Sample collection, macro- and micro- morphological examination Six fresh specimens of Paraisaria species were collected from insect hosts in An- hui, Guizhou and Yunnan Provinces, China. Detailed metadata, including geographic coordinates and collection sites, were recorded during fieldwork (Rathnayaka et al. 2025). Those samples were then transported to the laboratory in plastic containers for further examination. In the laboratory, fruiting bodies were sectioned and exam- ined using stereomicroscopes (Nikon SMZ 745 and SMZ 800N, Tokyo, Japan) to observe macroscopic features. Micromorphological traits, such as perithecia, asci, ascospores, synnemata, conidiophores, phialides and conidia, were documented using a Nikon DS-Ri2 digital camera attached to a Nikon ECLIPSE microscope, fol- lowing the methodology outlined by Senanayake et al. (2020). Isolation and material deposition A pure culture was obtained by transferring a small mass of mycelium from inside the host body to potato dextrose agar (PDA) with a flame-sterilised nee- dle under aseptic conditions, then incubated at 25 °C in the dark. The resulting strains were deposited in the Guizhou Culture Collection (GZCC), China and dried specimens were deposited at the Herbarium of Cryptogams, Kunming In- stitute of Botany, Academia Sinica (HKAS). Morphological data were analysed using the Tarosoft (R) v.0.9.7 Image Framework and photographic images were produced and edited using Adobe Photoshop CC 2022 (Adobe Systems, USA). To ensure accurate taxonomic documentation, Facesoffungi and Index Fun- gorum numbers were assigned to the newly-described species, following the guidelines of Jayasiri et al. (2015) and https://www.indexfungorum.org/. The introduction of new species followed the protocols established by Maharach- chikumbura et al. (2021) and Jayawardena et al. (2021). DNA extraction, PCR amplification and sequencing Fungal genomic DNA was extracted from both dried samples and cultures us- ing the E.Z.N.A.® Plant & Fungal DNA Kit (Omega Bio-Tek, USA) according to the manufacturer's protocol. The extracted DNA was stored at -20 °C for future use. Four gene regions, internal transcribed spacers (ITS), large subunit rDNA (LSU), small subunit rDNA (LSU), transcription elongation factor 1-alpha gene region (tef-1a), largest subunit of RNA polymerase II (rpb1) and RNA polymerase Il subunit (rpb2), were amplified and sequenced using primers listed in Table 1. PCR amplifications were performed in a 25 ul reaction volume containing 2 ul of DNA template, 8.5 ul of nuclease-free water, 1 pl of each primer (10 uM, final concentration 0.4 uM) and 12.5 ul of 2 x BenchTop™ Tag Master Mix (Biomiga, USA), which provides 1.25 units of Taq DNA polymerase per reaction. Primers were synthesised by Tsingke Biotech (Beijing, China). The PCR cycle included an initial denaturation at 98 °C for 2 minutes, followed by 40 cycles of 98 °C for 10 seconds, 55 °C for 1 minute and 72 °C for 30 seconds, with a final extension at 72 °C for 2 minutes. PCR products were examined by electrophoresis on a 1% (w/v) agarose gel in 1 x TAE buffer, stained with 4S Green Plus Nucleic Acid Stain (TSINGKE Biotech, China) and visualised under UV light. Agarose powder was MycoKeys 121: 253-270 (2025), DOI: 10.3897/mycokeys.121.156843 955 Yu Yang et al.: Three new species of Paraisaria (Ophiocordycipitaceae, Hypocreales) from China Table 1. Sequences of primers used in this study. Locus | Primers Primer sequence (5'-3’) References ITS ITS4 TCCTCCGCTTATTGATATGC White et al. (1990) ITS5 GGAAGTAAAAGTCGTAACAAGG SSU NS1 GTAGTCATATGCTTGTCTC White et al. (1990) NS4 CTTCCGTCAATTCCTTTAAG LSU LROR ACCCGCTGAACTTAAGC Vilgalys and Hester (1990) LR5 TCCTGAGGGAAACTTCG tef-la | EF1-983F | GCYCCYGGHCAYCGTGAYTTYAT | Carbone and Kohn (1999); Rehner and Buckley (2005) EF1-2218R | ATGACACCRACRGCRACRGTYTG rpb1 CRPB1A | CAYCCWGGYTTYATCAAGAA Castlebury et al. (2004) RPB1Cr_ | CCNGCDATNTCRTTRTCCATRTA rpb2. | ~fRPB2-5f | GAYGAYMGWGATCAYTTYGG Castlebury et al. (2004) fRPB2-7cR CCCATRGCTTGYTTRCCCAT purchased from Sangon Biotech (Shanghai, China) and sequences were obtained from Tsingke Biotechnology (Chongqing, China). Sequence assembly and editing were performed using BioEdit v.7.0.9 (Hall 1999). The resulting sequences were submitted to GenBank and their accession numbers are provided in Table 2. Phylogenetic analyses The newly-generated sequences were assembled using SeqMan version 11.1.0 (DNASTAR, Inc., Madison, WI, USA), while reference and closely-related taxa for phylogenetic analysis were selected through BLAST searches on NCBI Gen- Bank (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and by reviewing relevant litera- ture (Sung et al. 2007; Quandt et al. 2014; Ban et al. 2015; Sanjuan et al. 2015; Mongkolsamrit et al. 2019; Wei et al. 2021; Tehan et al. 2023) (Table 1). Phy- logenetic inference included both previously published and newly-generated sequences. Sequence alignment for each nuclear locus region was conducted using the ‘auto’ option in MAFFT (Katoh and Standley 2013), followed by refine- ment using the ‘gappyout’ approach in TrimAl (Capella-Gutiérrez et al. 2009). The most appropriate nucleotide substitution models for each dataset were selected using the Bayesian Information Criterion (BIC), derived from a set of 22 commonly used DNA substitution models that incorporate rate heterogene- ity, as implemented by ModelFinder (Kalyaanamoorthy et al. 2017). The aligned sequences were then concatenated and partitioning schemes were applied; further phylogenetic analysis was conducted. Maximum Likelihood (ML) analyses were conducted using RAXML-HPC2 (Stamatakis 2014) on the CIPRES Science Gateway V. 3.3 (Miller and Blair 2009), with default settings, except for 1,000 bootstrap replicates. For Bayesian Inference (BI), the GTR+I+G nucleotide substitution model was selected as the best-fit model using MrModelTest 2.2 (Nylander 2004) and posterior probabil- ities (PP) were estimated using Markov Chain Monte Carlo (MCMC) sampling in MrBayes v.3.1.2 (Ronquist et al. 2012). The BI analysis was conducted using six simultaneous Markov chains, with trees sampled every 100 generations and ran for 5,000,000 generations, stopping once the average standard deviation of split frequencies dropped below 0.01. Convergence was verified using TRAC- ER v.1.6 (Rambaut et al. 2013). The first 25% of the sampled trees were dis- carded as a burn-in period and the remaining trees were used to calculate PP. MycoKeys 121: 253-270 (2025), DOI: 10.3897/mycokeys.121.156843 256 Yu Yang et al.: Three new species of Paraisaria (Ophiocordycipitaceae, Hypocreales) from China Table 2. Names, strain numbers, references and corresponding GenBank accession numbers of the taxa used in the phylogenetic analyses of this study. Taxa names Paraisaria alba Paraisaria amazonica Paraisaria amazonica Paraisaria anhuiensis Paraisaria anhuiensis Paraisaria arcta Paraisaria arcta Paraisaria blattarioides Paraisaria blattarioides Paraisaria cascadensis Paraisaria cascadensis Paraisaria coenomyiae Paraisaria coenomyiae Paraisaria coleopterorum Paraisaria coleopterorum Paraisaria dubia Paraisaria gracilioides Paraisaria gracilioides Paraisaria gracilis Paraisaria gracilis Paraisaria heteropoda Paraisaria heteropoda Paraisaria heteropoda Paraisaria heteropoda Paraisaria insignis Paraisaria insignis Paraisaria monticola Paraisaria paramyrmicarum Paraisaria orthopterorum Paraisaria orthopterorum Paraisaria phuwiangensis Paraisaria phuwiangensis Paraisaria pseudoheteropoda Paraisaria pseudoheteropoda Paraisaria rosea Paraisaria sp. Paraisaria sp. Paraisaria tettigonia Paraisaria tettigoniae Paraisaria tettigoniae Paraisaria tettigoniae Paraisaria yodhathaii Paraisaria yodhathaii Tolypocladium inflatum Tolypocladium ophioglossoides GenBank accession numbers Specimen/ Strain number LSU ITS SSU tef-1a rpb1 rpb2 References HKAS 102484 | MN943839 | MN947219 | MN943843 | MN929085 | MN929078 | MN929082 Wei et al. (2021) HUA 186143. | KJ917571 - KJ917562 | KM411989 | KP212902 | KM411982 Sanjuan et al. (2015) HUA 186113 | KJ917572 - KJ917566 - KP212903 | KM411980 Sanjuan et al. (2015) HKAS 132203 | PV139238 | PV139207 | PV139224 | PV156001 | PV155972 | PV155987 This study HKAS 132204 | PV139239 | PV139208 | PV139225 | PV156002 | PV155973 | PV155988 This study HKAS 102553 | MN943841 | MN947221 | MN943845 | MN929087 | MN929080 - Wei et al. (2021) HKAS 102552 | MN943840 | MN947220 | MN943844 | MN929086 | MN929079 | MN929083 Wei et al. (2021) HUA186093 | KJ917570 - KJ917559 | KM411992 | KP212910 - Sanjuan et al. (2015) HUA 186108 | KJ917569 - KJ917558 - KP212912 | KM411984 Sanjuan et al. (2015) OSC-M-052010 | 0Q708931 | 0Q709237 | 0Q800918 | OR199814 | OR199828 | OR199838 Tehan et al. (2023) OSC-M-052017 | 0Q708934 | 0Q709240 | 0Q800921 | OR199817 | OR199831 - Tehan et al. (2023) NBRC 106964 | AB968413 | AB968397 | AB968385 | AB968571 ~ AB968533 Ban et al. (2015) NBRC 108993 | AB968412 | AB968396 | AB968384 | AB968570 - AB968532 Ban et al. (2015) HKAS 145895 | PV139240 | PV139209 - PV156003 | PV155974 | PV155989 This study HKAS 145894 | PV139241 | PV139210 - PV156004 | PV155975 | PV155990 This study NJU985 - - MT918426 - - - Yuan et al. (2022) HUA186095 - - KJ917556 | KM411994 | KP212914 - Sanjuan et al. (2015) HUA 186092 | KJ130992 - KJ917555 - KP212915 - Sanjuan et al. (2015) EFCC 3101 | EF468810 - EF468955 | EF468750 | EF468858 | EF468913 Sung et al. (2007) EFCC 8572 | EF468811 | JNO49851 | EF468956 | EF468751 | EF468859 | EF468912 Sung et al. (2007) OSC 106404 | AY489722 - AY489690 | AY489617 | AY489651 - Quandt et al. (2014) EFCC 10125 | EF468812 | JNO49852 | EF468957 | EF468752 | EF468860 | EF468914 Sung et al. (2007) NBRC 100643 | JN941422 - JN941719 | AB968595 | JN992453 | AB968556 Ban et al. (2015) BCC 18246 - AB968411 | AB113352 | MK214083 | MK214087 - Mongkolsamrit et al. (2019) OSC-M-052013 | 0Q708938 | 0Q709244 | 0Q800924 | OR199820 | 0R199834 - Tehan et al. (2023) OSC-M-052004 | 0Q708927 | 0Q709234 | 0Q800914 | 0R199810 - - Tehan et al. (2023) BP! 634610 - 0Q709246 - - - - Tehan et al. (2023) IMI 393961 | EU797600 - - EU797597 - - Evans et al. (2010) BBC 88305 | MK332583 | MH754742 - MK214080 | MK214084 - Mongkolsamrit et al. (2019) TBRC 9710 | MK332582 | MH754743 - MK214081 | MK214085 - Mongkolsamrit et al. (2019) TBRC 9709 | MK192057 | MK192015 - MK214082 | MK214086 ~ Mongkolsamrit et al. (2019) BBH 43491 | MK192058 | MH188542 ~ - MH211351 ~ Mongkolsamrit et al. (2019) OSC-M-052022 | 0Q708939 | 0Q709245 | 0Q800925 | OR199821 | OR199835 | OR199841 Tehan et al. (2023) OSC-M-052009 | 0Q708935 | 0Q709241 | 0Q800922 | 0R199818 | OR199832 | 0R199840 Tehan et al. (2023) HKAS_102546 | MN943842 | MN947222 | MN943846 | MN929088 | MN929081 | MN929084 Wei et al. (2021) OSC-M-052011 | 0Q708932 | 0Q709238 | 0Q800919 | OR199815 | OR199829 | OR199839 Tehan et al. (2023) OSC-M-052026 | 0Q708936 | 0Q709242 - - - - Tehan et al. (2023) GZUH - - KT345955 | KT375440 | KT375441 - Wen et al. (2016) CS14062709 HKAS 144580 | PV139242 | PV139211 | PV139226 | PV156005 | PV155976 | PV155991 This study HKAS 132245 | PV139244 | PV139213 | PV139228 | PV156007 | PV155978 | PV155993 This study GZCC 24-0222 | PV139243 | PV139212 | PV139227 | PV156006 | PV155977 | PV155992 This study BBH 43163 | MK332584 | MH188539 - MH211353 | MH211349 - Mongkolsamrrit et al. (2019) TBRC 8502. | MH201168 | MH188540 - MH211354 | MH211350 — Mongkolsamrit et al. (2019) OSC 71235 | EF469077 | JNO49844 | EF469124 | EF469061 | EF469090 | EF469108 Sung et al. (2007) NBRC 106332 | JN941409 | JN943322 | JN941732 - JN992466 | MN929082 Schoch et al. (2012) Note: The symbol “—” means that the sequence is not available and newly-generated sequences in this study are in bold. MycoKeys 121: 253-270 (2025), DOI: 10.3897/mycokeys.121.156843 Tolypocladium inflatum (OSC 71235) and T. ophioglossoides (NBRC 106332) were chosen as outgroups. Significant support was determined as ML boot- strap values = 75% and BI posterior probabilities = 0.90. The final phylogenetic tree was visualised using FigTree v.1.4.0 (Rambaut 2012). 257 Yu Yang et al.: Three new species of Paraisaria (Ophiocordycipitaceae, Hypocreales) from China Phylogenetic analysis results The dataset combined LSU, ITS, SSU, tef-1a, rpb1 and rpb2 sequence data and encompassed 45 strains representing 26 taxa, with Tolypocladium inflatum (OSC 71235) and T. ophioglossoides (NBRC 106332) as outgroup taxa. It included 4845 aligned characters, distributed as follows: LSU (1-838 bp), ITS (839-1360 bp), SSU (1361-2343 bp), tef-1a (2344-3230 bp), rpbb1 (3231-3885 bp) and rpb2 (3886-4845 bp). The tree topology of the RAXML analysis was consistent with that of the Bayesian analysis. The best-scoring RAXML tree had a final likelihood value of -16818.111708 (Fig. 1). The estimated base frequencies were A = 0.234476, Paraisaria tettigonia HKAS 144580 Orthoptera Paraisaria tettigonia GZCC 24-0222 95/0.981 Paraisaria tettigonia HKAS 132245 Paraisaria tettigonia GZUH CS14062709 100/0.99 | ;Paraisaria gracilis EFCC 8572 Lenidunters 75) © Paraisaria gracilis EFCC 3101 prop , gal100/1{ Paraisaria orthopterorum BBC 88305 eat Paraisaria orthopterorum TBRC 9710 Orthoptera hee 77.\_\Paraisaria coleopterorum HKAS 145895 Paraisaria coleopterorum HKAS 145894 Coleoptera 99/0.99 |Paraisaria phuwiangensis TBRC 9709 99/0.94] ‘Paraisaria phuwiangensis BBH 43491 Paraisaria monticola BPI 634610 Orthoptera Paraisaria yodhathaii BBH 43163 Paraisaria yodhathaii TBRC 8502 79/1 -Paraisaria alba HKAS 102484 Paraisaria amazonica HUA 186143 Orthoptera ane Paraisaria amazonica HUA 186113 100/17 Paraisaria blattarioides HUA186093 100/0.94 Coleoptera 99.8/100 100/1_|l|,.,4, “Paraisaria blattarioides HUA 186108 nant let b9/1|Paraisaria anhuiensis HKAS 132203 bdo gg araisaria anhuiensis HKAS 132204 100/1 Paraisaria rosea HKAS 102546 c A Ne oleoptera Paraisaria gracilioides HUA 186092 100/1' Pargisaria gracilioides HUA186095 100/1_|Paraisaria arcta HKAS 102553 ; Paraisaria arcta HKAS 102552 Lepidoptera Paraisaria sp. OSC M 052011 Insecta 84/-| 'Paraisaria dubia NJU985 Lepidoptera 82/, Paraisaria paramyrmicarum |MI 393961 Hymenoptera 100/1 98/0.96'— Paraisaria sp. OSC M 052026 Insecta agi] Paraisaria cascadensis OSC M 052010 100 Paraisaria cascadensis OSC M 052017 Orthoptera oof Paraisaria heteropoda BCC 18246 ; g2/,__ Paraisaria heteropoda NBRC 100643 Hemiptera aoa 100/17 Paraisaria insignis OSC M 052013 Paraisaria insignis OSC M 052004 Coleoptera 99/1] Paraisaria pseudoheteropoda OSC M 052022 aA 9940.97 * Paraisaria pseudoheteropoda OSC M 052009 Hemiptera g9/-_ Paraisaria heteropoda OSC 106404 100/1| Paraisaria coenomyiae NBRC 106964 : Paraisaria coenomyiae NBRC 108993 Diptera Paraisaria heteropoda EFCC 10125 Hemiptera 100/1 Tolypocladium inflatum OSC 71235 0.02 Tolypocladium ophioglossoides NBRC 106332 Out group Figure 1. A phylogenetic tree was constructed using Maximum Likelihood (ML) analysis in RAxML, incorporating se- quence data from multi-nuclear loci regions: LSU, ITS, SSU, tef-1a, rpbb1 and rpb2. The analysis included Tolypocladium inflatum and T. ophioglossoides as outgroup taxa. Significant nodes, with ML bootstrap values equal to or greater than 75% and Bayesian posterior probabilities equal to or greater than 0.90, are indicated on the phylogram. Newly-generated sequences are emphasised in bold red for clarity. MycoKeys 121: 253-270 (2025), DOI: 10.3897/mycokeys.121.156843 959 Yu Yang et al.: Three new species of Paraisaria (Ophiocordycipitaceae, Hypocreales) from China C = 0.281939, G = 0.285069, T = 0.198516, with substitution rates as follows: AC = 1.237820, AG = 3.925347, AT = 0.890655, CG = 1.291471, CT = 7.097502, GT = 1.000000. The gamma distribution shape parameter a = 0.795567. The to- pologies from both the Maximum Likelihood (ML) and Bayesian analyses were manually reviewed and showed substantial agreement. Based on the phylogenet- ic results, two new species were recognised: Paraisaria anhuiensis, P coleoptero- rum and one asexual morph record of Paraisaria tettigoniae. Taxonomy Paraisaria anhuiensis Y. P. Xiao, K.D. Hyde & Y. Yang, sp. nov. Index Fungorum: IF903775 Facesoffungi Number: FoF17627 Fig. 2 Etymology. The epithet “anhuiensis” refers to the type location “Anhui Prov- ince, China”. Holotype. CHINA * Anhui Province, Chuzhou City, occurs on the larvae of Co- leoptera, on leaf litter, 191 m elev., 118.05°E, 32.33°N, 25 August 2021, Yu Yang, HFS29 (HKAS 132203, holotype). Description. Parasitic on the larvae of Coleoptera. Host 2.1-3.3 long x 0.2-0.4 cm wide, reddish-brown, without hyphae on the surface. Sexu- al morph: Stromata 2.4-—3.5 x 0.18-0.32 cm diam., mostly single, cylindrical, unbranched, emerging from the head of the larva body, yellowish-white. Fer- tile head 4.5 x 6 mm, subglobose, pale yellow when fresh, pale yellow-brown when dry, distinct from the stipe. Stipe 1.5-—2.3 x 0.18-0.21 cm, pale yellow, straight, unbranched, glossy, cylindrical, inside not hollow. Perithecia 639-786 x 139-201 um (X = 712.5 x 170 um, n = 30), completely immersed, ampulliform, ostiolate, thick-walled. Asci 371-480 x 6.6-7.2 um (x = 425.5 x 6.9 um, n = 30), hyaline, filiform, with a thin apex. Apical cap 5.7-6.6 x 3.1-4.5 um (x = 5.4 x 3.4 um, n = 40), with a small channel in the centre. Ascospores filiform, equal to the asci in length, when mature, breaking into numerous secondary asco- spores. Secondary ascospores 6.3-9.1 x 1.3-1.9 um (xX = 7.7 x 1.6, n = 40), cylindrical, one-celled, straight, hyaline, smooth. Asexual morph Not observed. Other material examined. CHINA * Anhui Province, Huangshan City, parasitic on larvae of Coleoptera, on the soil, 403 m elev., 117.48E, 30.22N, 9 August 2023, Yu Yang, AH23190 (HKAS 132204, paratype). Notes. The multi-locus phylogenetic analysis revealed that Paraisaria anhui- ensis clusters with P. rosea, with 99% MLBP and 0.95 PP statistical support (Fig. 1). Morphologically, Paraisaria anhuiensis differs from P. rosea by produc- ing longer asci (371-480 x 6.6—7.2 um vs. 230-390 x 3.5—6 um; L/W ratio 61.7 vs. 65.3) (Wei et al. 2021). Paraisaria anhuiensis differs from P. rosea in that its stromata emerge from the larval head and feature a pale-yellow fertile head, whereas P rosea produces stromata from the middle part of the larval body, with a pink fertile head (Wei et al. 2021). Pairwise sequence comparison shows 2.16% (10/463 bp) in ITS, 0.58% (5/850) in tef-1a, 1.58% (10/632 bp) in rpb1 and 1.71% (17 out of 992 bp) in rpb2 between P. anhuiensis and P. rosea (Wei et al. 2021). Hence, we describe Paraisaria anhuiensis as a new species, based on its distinctive morphology and molecular evidence. MycoKeys 121: 253-270 (2025), DOI: 10.3897/mycokeys.121.156843 959 Yu Yang et al.: Three new species of Paraisaria (Ophiocordycipitaceae, Hypocreales) from China : Figure 2. Paraisaria anhuiensis (HKAS 132203, holotype) a. Habitat; b. Overview of the host and stromata; c. Fertile head; d. Host; e, f. Vertical section of ascostroma; g-i. Asci; j. Apical cap; k-m. Secondary ascospores. Scale bars: 500 um (f); 200 um (g); 100 um (h); 50 um (i); 5 um (j-m). MycoKeys 121: 253-270 (2025), DOI: 10.3897/mycokeys.121.156843 260 Yu Yang et al.: Three new species of Paraisaria (Ophiocordycipitaceae, Hypocreales) from China Paraisaria coleopterorum Y. Yang, K.D. Hyde & Y. P. Xiao, sp. nov. Index Fungorum: IF903776 Facesoffungi Number: FoF 17628 Fig. 3 Etymology. The epithet “co/eopterorum” refers to its host belonging to the Co- leoptera larvae. Holotype. CHINA * Yunnan Province, Honghe Hani and Yi Autonomous Prefec- ture, Honghe County, parasitic on larva of Coleoptera, buried in the soil, 1963 m elev., 102.291E, 23.271N, 18 July 2024, Yu Yang, YY24340 (HKAS 145895, holotype) Description. Parasitic on a larva of Coleoptera. Host 1.5-2.8 long x 0.3-0.5 cm wide, bark brown, without hyphae on the surface. Sexual morph Stromata 2.4—4.5 x 0.2-0.4 cm, typically solitary, cylindrical, unbranched, emerging from the larval body, simple, erect, pale yellowish-brown. Fertile head 4.5 x 5.6 mm, subglobose, pale yellowish-brown at the apex, becoming paler towards the base when fresh, turning pale pink when dry, distinctly separate from the stipe. Stipe 1.8-4 x 0.12- 0.23 cm, pale yellow, straight, unbranched, glossy, cylindrical, with a solid interior. Perithecia 620-680 x 110-156 um (xX = 650 x 133 um, n = 30), completely im- mersed, thick-walled. Peridium 22-36 (x = 29, n = 30) um wide, comprising hya- line, three layers, textura porrecta outer layer forming a dense palisade layer cover- ing the fertile head, textura intricata middle layer, textura porrecta inner layer. Asci 510-590 x 4.6-6.2 um (X = 550 x 5.4 um, n = 30), hyaline, cylindrical, with a thin apex. Apical cap 6.3-7.1 x 3.1—4.1 um (X = 6.7 x 3.6 um, n = 40). Ascospores equal in length to the asci, fragmenting into numerous secondary ascospores upon ma- turity. Secondary ascospores 6.4—-8.1 x 1.6-2.3 um (xX = 7.2 x 1.9 um, n = 40), cylindrical, one-celled, hyaline and smooth-walled. Asexual morph Not observed. Other material examined. CHINA * Yunnan Province, Honghe Hani and Yi Au- tonomous Prefecture, Honghe County, parasitic on a larva of Coleoptera, buried in the soil, 1963 m elev., 102.291E, 23.271N, 18 July 2024, Yu Yang, YY24343 (HKAS 145894, paratype). Notes. Paraisaria coleopterorum clustered with P gracilis, RP orthopterorum and P. phuwiangensis in the phylogenetic tree with 88% MLBP, 0.98 PP support (Fig. 1). Pairwise sequence comparisons revealed differences of 1.47—2.75% (8-15/545) in ITS, 0.95-1.19% (8-10/836) in LSU, 0.76-1.30% (7- 12/923) in tef- 1a and 1.06-1.48% (10-14/943) in rpbb1 between P. coleopterorum and P. grac- ilis/P. orthopterorum/P. phuwiangensis, respectively. The host of P. coleoptero- rum is the larva of Coleoptera, while P orthopterorum infects Orthoptera nymphs (Mongkolsamrit et al. 2019). Compared to P orthopterorum, P. coleopterorum produces longer and thinner perithecia (620-680 x 110-156 um vs. 520-650 x 150-250 um); L/W ratio 4.9 vs. 2.9) and longer asci (510-590 x 4.6-6.2 um vs. 400 x 5 um; L/W ratio 101.9 vs. 80) (Mongkolsamrit et al. 2019). When com- pared to P phuwiangensis, P coleopterorum has smaller perithecia (620-680 x 110-156 um vs. 800-1200 x 300-380 um; L/W ratio 4.9 vs. 2.9) and longer asci (510-590 x 4.6-6.2 um vs. 500 x 3-5 um; L/W ratio 101.9 vs. 125). Compared to P gracilis, P coleopterorum produces smaller perithecia (620-680 x 110-156 um vs. 560-840 x 200-360 um); L/W ratio 4.9 vs. 2.5) and longer asci (510-590 x 4.6-6.2 um vs. 400-528 x 5-8 um; L/W ratio 101.9 vs. 71.4) (Mongkolsamrit et al. 2019). Therefore, both morphological and phylogenetic analyses support the distinction of P coleopterorum as a new species in Paraisaria. MycoKeys 121: 253-270 (2025), DOI: 10.3897/mycokeys.121.156843 761 Yu Yang et al.: Three new species of Paraisaria (Ophiocordycipitaceae, Hypocreales) from China J + ‘ 1 : ‘ =| : 4 > -—, — —) —, —— eae T ane. t leis! ao -—| ~- = amy =] "| | | 4 & | =| ’ mq | i Figure 3. Paraisaria coleopterorum (HKAS 145895, holotype) a. Habitat; b. Overview of the host and stromata; c. Host; d. Fertile head; e. Vertical section of ascostroma; f. Peridium; g-j. Asci; k. Apical cap; |. Secondary ascospores. Scale bars: 200 um (e); 50 um (f); 100 um (g-j); 20 um (k); 5 um (I). MycoKeys 121: 253-270 (2025), DOI: 10.3897/mycokeys.121.156843 262 Yu Yang et al.: Three new species of Paraisaria (Ophiocordycipitaceae, Hypocreales) from China Paraisaria tettigoniae (T.C. Wen, Y.P. Xiao & K.D. Hyde) Luangsa-ard, Mongkols. & Samson [as ‘tettigonia’], Mycol. Progr. 18(9): 1225 (2019) Index Fungorum: IF839725 Fig. 4 Description. Parasitic on adults of Orthoptera, found on the leaf litter. Host measuring 1.5-2.8 cm long, 5-8 mm wide, with hyphae present on the sur- face. Sexual morph Stromata 1.2-2.5 cm long, 2-5 mm wide, arising singly or in groups from the host prothorax stipitate, capitate, unbranched, yellow- ish-white to pale yellow when fresh, turning yellowish-brown when dry. Stipe 1-3.5 cm long, 1.2-1.8 mm diameter, yellowish white to pale yellow, cylindrical in shape, terminating in a fertile apex. Fertile head globoid, 1.5—4 mm, pale yel- low and solitary. Perithecia 500-630 x 180-220 um (x= 565 x 200 um, n = 30), immersed, ovoid to flask-shaped, thick-walled. Asci 243-310 x 4.3-6.5 um (X= 276 x 5.4 um, n = 50), hyaline, cylindrical, with a thickened apex. Apical cap 4.9-6.7 x 3.4-4.4 um (X= 5.8 x 3.9 um, n = 50), thick, hyaline. Ascospores cylin- drical, hyaline, as long as the asci, fragmenting into part-spores. Secondary as- cospores 6.1—8.5 x 1.8-2.5 um (X= 7.4 x 1.8 um, n = 50) cylindrical, one-celled, straight, hyaline, smooth-walled. Asexual morph Hyphomycetous. Synnemata emerge from the insect body, white, 2-4 mm long, 0.2-—0.5 mm wide. Conidio- phores 21-43 um long (x = 32 um, n = 30), irregularly differentiated from the synnemata, sparse, gregarious and branched. Phialides 12-17 x 2.3-4.8 um (x= 14.5 x 3.5 um, n = 25), cylindrical with 1-3 necks, hyaline, aseptate, phia- lidic. Conidia 4.2—5.8 x 1.2-1.8 um (x = 5 x 1.5 um, n = 30), solitary, hyaline, aseptate, cylindrical with rounded tips, smooth-walled. Culture characteristics. Colonies on PDA medium grow slowly, isolated from tissue taken inside the host body and are circular, reaching 2 cm in diam- eter after 35 days at 25 °C, with a white appearance. Conidiophore 50-90 um (x = 70 um, n = 40), bearing 2-4 phialides in one. Phialides 20-50 x 2.1-5.2 um (x = 35 x 3.6 um, n = 40) solitary, arising laterally from hyphae, hyaline, smooth. Conidia 7.8-14.5 x 2.1-3.2 um (X = 11.1 x 2.6 um, n = 40) hyaline, unicellular, ellipsoid, some slightly curved, smooth-walled. Material examined. CHINA * Guizhou Province, Guiyang City, Xiuwen County, at 709 m elev., 27.256N, 106.674E, parasitic on adult of Orthoptera, collected on the leaf litter, 30 May 2024, Yu Yang XW2416 (HKAS 144580). XW2416J (GZCC 24- 0222, living culture); China * Guizhou Province, Qiandongnan Miao and Dong Au- tonomous Prefecture, Zhen Yuan County, at 555 m elev., 27.111N, 108.401E, par- asitic on an adult of Orthoptera, 26 June 2023, Yu Yang, TX23108 (HKAS 132245). Notes. Our new collection is phylogenetically closely related to Paraisaria tet- tigoniae, with 95% MLBP and 0.98 PP support (Fig. 1). It shares highly similar sequences with P tettigoniae across multiple loci (SSU, tef-1a and rpb1), where- as P tettigoniae shows anomalous divergence. Further inspection indicates that the ITS sequence of P tettigoniae (GenBank accession: KT345954) may be prob- lematic, possibly due to sequencing errors or intragenomic variation. Therefore, this ITS sequence (strain GZUH CS14062709) was excluded from our analysis. Paraisaria tettigoniae was originally described from Guizhou, China, based on its sexual morph parasitising adult Orthoptera (Wen et al. 2016). Our collection, also from Guizhou, represents the asexual morph of the same species. Although minor morphological differences were observed — such as smaller perithecia MycoKeys 121: 253-270 (2025), DOI: 10.3897/mycokeys.121.156843 763 Yu Yang et al.: Three new species of Paraisaria (Ophiocordycipitaceae, Hypocreales) from China Figure 4. Paraisaria tettigoniae (HKAS 144580 and HKAS 132245). a. Habitat; b. Overview of the host and stromata; c. Stromata; d. Perithecia; e-g. Asci; h. Apical cap; i, j. Secondary ascospores; k. Synnemata on host; |. Synnemata; m, n. Phialides; p. Conidia; q. Culture; r, s. Conidiophores; t. Conidia on the phialides; u. Conidia. Scale bars: 500 um (d); 100 um (e-g); 50 um (I, r-s); 10 um (h-j, m-o, t-u); 5 ym (p). MycoKeys 121: 253-270 (2025), DOI: 10.3897/mycokeys.121.156843 264 Yu Yang et al.: Three new species of Paraisaria (Ophiocordycipitaceae, Hypocreales) from China (500-630 x 180-220 um vs. 520-680 x 205-275 um) and shorter asci (243- 310 x 4.3-6.5 um vs. 530-615 x 6.5-9.3 um) — the multi-locus phylogenetic analysis (excluding the problematic ITS sequence) shows that our collection clusters with P. tettigoniae (Fig. 1). Therefore, based on both morphological characteristics and multi-locus phylogenetic evidence, our collection is identi- fied as Paraisaria tettigoniae, representing the first report of its asexual morph. Discussion Taxonomic studies on Paraisaria in China remain underexplored compared to other fungal groups (Yang et al. 2021, 2024; Xiao et al. 2023, 2024). Un- til the present study, only three Paraisaria new species have been described from China, based on morphological and molecular evidence (Wen et al. 2016; Wei et al. 2021). Wen et al. (2016) introduced Ophiocordyceps tettigonia from Guizhou Province, China, which was subsequently transferred to Paraisaria, based on multi-gene phylogenetic analysis and morphological characterisa- tion (Wen et al. 2016; Mongkolsamrit et al. 2019). Subsequently, Wei et al. (2021) described Paraisaria arcta from Guizhou Province and P. rosea from Yunnan Province. These findings highlight the limited exploration of the taxo- nomic diversity of the genus in China. We describe two new species of Para- isaria (P anhuiensis and P. coleopterorum) and report one asexual morph of Paraisaria tettigoniae using an integrative approach that combines morpholog- ical characteristics with phylogenetic analyses. These newly-recognised taxa, including two new species and one asexual morph record, are each placed in well-supported clades within the phylogenetic tree (Fig. 1). Paraisaria fungi exhibit remarkable parasitic versatility, infecting a di- verse range of insect hosts across multiple orders, including Coleoptera, Dictyoptera, Diptera, Lepidoptera, Orthoptera, Hemiptera and Hymenoptera (Kobayasi 1941; Evans et al. 2010; Mongkolsamrit et al. 2019; Wei et al. 2021; Tehan et al. 2023). Most species in this genus predominantly parasit- ise Orthoptera and Coleoptera, as reflected in the phylogenetic tree (Fig. 1). P. anhuiensis and P. coleopterorum form well-supported, separate clades in the phylogenetic tree (highlighted in red; Fig. 1), demonstrating their molec- ular distinctiveness. These placements, together with their diagnostic mor- phological differences, provide robust support for their recognition as inde- pendent species. Furthermore, detailed morphological comparisons reveal significant diagnostic differences, which reinforce their unique taxonomic identities and support their classification as distinct species. In addition, the asexual morph of P tettigoniae is reported here for the first time, enrich- ing our understanding of its life cycle and expanding the known morpholog- ical diversity within Paraisaria. The discovery of two new species and the report of one asexual morph re- cord in this study significantly expands our understanding of the taxonomic diversity within Paraisaria. Previous studies have highlighted the ecological and economic importance of the genus, including its anti-oxidative and antibacteri- al properties (Ma et al. 2012; Suo et al. 2014; Huang et al. 2019), as well as its potential risks to public health due to mycotoxin production (Doan et al. 2017; Tehan et al. 2023). However, the taxonomic boundaries and species diversity of Paraisaria have remained poorly resolved, hindering further exploration of MycoKeys 121: 253-270 (2025), DOI: 10.3897/mycokeys.121.156843 065 Yu Yang et al.: Three new species of Paraisaria (Ophiocordycipitaceae, Hypocreales) from China its potential applications. Our findings provide a foundation for future studies to investigate the ecological roles, chemical diversity and functional genomics of these newly-described species. Future studies could integrate multi-omics approaches, such as metabolomics and transcriptomics, to further explore the ecological roles and functional potential of Paraisaria. Acknowledgements Yu Yang would like to thank the Mushroom Research Foundation, Chiang Rai, Thailand, for supporting this research. Ruvishika S. Jayawardena would like to thank the Eminent Scholar offered by Kyun Hee University. The authors would also like to thank Shaun Pennycook (Manaaki Whenua Landcare Research, New Zealand) for advising on fungal nomenclature. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Use of Al No use of Al was reported. Funding This work was funded by Guizhou Provincial Basic Research Program (MS [2025] No. 193), the Science and Technology Foundation of Guizhou Province (Qian Ke He Pingtai ZSYS[2025]029, Guizhou Provincial Science and Technology Department (KXJZ[2024]021) and the High-level Talent Research Initiation Fund Project in Guizhou Institute of Technology (2023GCC063). The work was also funded by the Guizhou In- stitute of Technology 2024 Academic New Bud Cultivation and Innovation Explora- tion Project (No. 2024XSXMO08) and the Distinguished Scientist Fellowship Program (DSFP), King Saud University, Kingdom of Saudi Arabia. Author contributions Methodology: SQX, XDL. Writing - original draft: . Writing - review and editing: YZL, KDH, RSJ, AM, YX, SN. Author ORCIDs Yu Yang ® https://orcid.org/0000-0001-8268-487X Kevin D. Hyde © https://orcid.org/0000-0002-2191-0762 Ausana Mapook © https://orcid.org/0000-0001-7929-2429 Yong-Zhong Lu ® https://orcid.org/0000-0002-1033-5782 Somrudee Nilthong © https://orcid.org/0000-0002-7454-5826 Shu-Qiong Xie © https://orcid.org/0009-0006-801 1-7680 Xiang-Dong Li © https://orcid.org/0009-0001-4404-1803 Ruvishika S. Jayawardena © https://orcid.org/0000-0001-7702-4885 Yuan-Pin Xiao © https://orcid.org/0000-0003-1730-3545 MycoKeys 121: 253-270 (2025), DOI: 10.3897/mycokeys.121.156843 266 Yu Yang et al.: Three new species of Paraisaria (Ophiocordycipitaceae, Hypocreales) from China Data availability All of the data that support the findings of this study are available in the main text or Supplementary Information. 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Biodiver- sity Data Journal 9(e66115): 1-13. https://doi.org/10.3897/BDJ.9.e66115 Yang Y, Jayawardena Ruvishika S, Lu YZ, Xie SQ, Tian XG, Wang JP, Zhou SX, Xiao YP (2024) Four new Ophiocordyceps species in China. Mycosystema 43(3): 230-256. https://doi.org/10.13346/j.mycosystema.230256 Yuan L, Tong L, Wang Y, Du Y, Liu M, He S, Wei S, Zhang Y, Chen Z, Jin S, Guo D (2022) Enhancing polysaccharide production by Paraisaria dubia spores batch fermentation through a pH-shift strategy based on kinetic analysis. Process Biochemistry 126: 292-298. https://doi.org/10.1016/j.procbio.2022.12.020 Supplementary material 1 Supplementary images Authors: Yu Yang, Kevin D. Hyde, Ausana Mapook, Yong-Zhong Lu, Somrudee Nilthong, Shu-Qiong Xie, Xiang-Dong Li, Ruvishika S. Jayawardena, Yuan-Pin Xiao Data type: docx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.121.156843.suppl1 MycoKeys 121: 253-270 (2025), DOI: 10.3897/mycokeys.121.156843 970