<8) MycoKeys MycoKeys 112: 335-359 (2025) DOI: 10.3897/mycokeys.112.140799 Research Article Four new araneogenous species and a new genus in Hypocreales (Clavicipitaceae, Cordycipitaceae) from the karst region of China Wan-Hao Chen'22®, Dan Li'4®, Hui-Lin Shu'’“®, Jian-Dong Liang'*®, Jie-Hong Zhao'™®, Wei-Yi Tian'2®, Yan-Feng Han2® Fe wo ND — Center for Mycomedicine Research, Basic Medical School, Guizhou University of Traditional Chinese Medicine, Guiyang 550025, Guizhou Province, China Institute of Fungus Resources, Department of Ecology, College of Life Sciences, Guizhou University, Guiyang 550025, Guizhou Province, China Key Laboratory of Microbio and Infectious Disease Prevention & Control in Guizhou Province, Guiyang 550025, Guizhou Province, China College of Pharmacy, Guizhou University of Traditional Chinese Medicine, Guiyang 550025, Guizhou Province, China Corresponding author: Yan-Feng Han (swallow1128@126.com) OPEN Qaceess Academic editor: Nalin Wijayawardene Received: 2 November 2024 Accepted: 22 December 2024 Published: 23 January 2025 Citation: Chen W-H, Li D, Shu H-L, Liang J-D, Zhao J-H, Tian W-Y, Han Y-F (2025) Four new araneogenous species and a new genus in Hypocreales (Clavicipitaceae, Cordycipitaceae) from the karst region of China. MycoKeys 112: 335-359. https://doi.org/10.3897/ mycokeys.112.140799 Copyright: © Wan-Hao Chen 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 The karst region in southwestern China is one of the biodiversity hotspots in the world with rich fungal diversity but under-studied. Four fungal species belonging to Chlorocil- lium (Clavicipitaceae) and Gamszarella (Cordycipitaceae) were isolated from dead spi- ders. Morphological comparisons, phylogenetic analyses and a PHI analysis based on multigene datasets support the establishment of these new species viz., Chlorocillium guizhouense sp. nov., C. vallense sp. nov., Gamszarella sinensis sp. nov., and G. vallensis sp. nov. are introduced. A new genus, Neogamszarella, is proposed to accommodate Gamszarella antillana, which is phylogenetically distinct from Gamszarella s. str. Our results revealed that further attention needs to be paid to the diversity of araneogenous fungi in the karst regions of southwestern China. Key words: Chlorocillium, lecanicillium-like, morphology, phylogenetic analysis, spider Introduction Araneogenous or araneopathogenic fungi are spider-pathogenic fungi and are one of the ecologically important groups of fungi (Evans and Samson 1987). Formerly, araneogenous fungi were restricted to three families in Hypocreales: Cordycipitaceae (Kobayasi and Shimizu 1982; Chen et al. 2018; Mongkolsamrit et al. 2018) and Ophiocordycipitaceae (Kobayasi 1941; Samson and Evans 1975), and one species in Bionectriaceae (Chen et al. 2016a; Shrestha et al. 2019). How- ever, Chen et al. (2022a) reported two new spider-associated genera in Clavic- ipitaceae. Wang et al. (2024a) reported Akanthomyces Lebert, Beauveria Vuill., Cordyceps Fr., Engyodontium de Hoog, Gibellula Cavara, Hevansia Luangsa-ard et al., Lecanicillium W. Gams & Zare, Samsoniella Mongkols. et al., Torrubiella Boud., Jenniferia Mongkols. et al., Polystromomyces Mongkols. et al., Bhushaniella Mongkolsamnrit et al., Hirsutella Pat., Hymenostilbe Petch, Ophiocordyceps Petch, Purpureocillium Luangsa-ard et al., Clonostachys Corda and Chlorocillium Zare & W. Gams, from spiders. Crous et al. (2023) introduced a new araneogenous genus, Gamszarella Crous, while Khonsanit et al. (2024) introduced two new 335 Wan-Hao Chen et al.: Four new araneogenous species and a new genus in Hypocreales spider-host genera, Arachnidicola Khons. et al., and Corniculantispora Khons., et al. and restricted the hosts of Akanthomyces to moths. Thus, the araneogenous fungi were distributed in 21 genera of Hypocreales (List of the genera see Suppl. material 1) (Crous et al. 2023; Khonsanit et al. 2024; Wang et al. 2024a). The karst region in southwestern China is one of the 36 biodiversity hotspots in the world (Delgado-Baquerizo et al. 2020). A large range of continuously distribut- ed primary forests exist in the karst region, with an exceptionally diverse ecosys- tem. As a result of the complex ecological environment and unique geographic conditions in this region, unique species can be described (Ozkan et al. 2010; Su et al. 2017). Seventeen new araneogenous species and new records were report- ed by Chen et al. (2012, 2016a, b, 2017a, b, 2018, 2019a, 2022a, b, c, 2023, 2024), Han et al. (2013) and Zou et al. (2016, 2021) from karst region of southwest China. During a survey of araneogenous fungi and their allies in southwestern Chi- na, infected spider specimens were collected, and fungal strains were isolated and purified. Isolated strains were identified based on the multigene phylogeny and morphological characteristics, and introduced four new species in Clavi- cipitaceae and Cordycipitaceae i.e. Chlorocillium guizhouense sp. nov., C. val- lense sp. nov., Gamszarella sinensis sp. nov., and G. vallensis sp. nov. Moreover, Gamszarella antillana is not congeneric with Gamszarella s. str. in the phyloge- netic analyses, thus, Neogamszarella is proposed to accommodate it. Materials and methods Specimen collection and identification The specimens were collected from Dali Dong Village (26°01'58.70'N, 108°24'48.06"E), Rongjiang County, Qiandongnan Miao and Dong Autonomous Prefecture, Mayao River Valley (26°21'24.71"N, 107°22'48.22"E), Duyun City, Qi- annan Buyi and Miao Autonomous Prefecture and Bala Valley (26°45'7.0344'N, 106°58'57.09"E), Wudang District, Guiyang, Guizhou Province, on 1%* October 2018, 4° September 2021 and 5" April 2024, respectively. The samples were placed in an ice box and brought to the laboratory. Specimens were preserved in the refrigerator at 4 °C until further processing. The surface of each arthro- pod body was rinsed with sterile water, followed by sterilization with 75% etha- nol for 3-5 s and rinsing again three times with sterilized water. After drying on sterilized filter paper, a piece of the synnema, mycelium or sclerotium was cut from the specimen and placed on plates of potato dextrose agar (PDA) or PDA modified by the addition of 1% w/v peptone containing 0.1 g/l streptomycin and 0.05 g/I tetracycline (Chen et al. 2019b). After fungal colonies emerged from the plated samples, a piece of mycelium from the colony edge was transferred onto new agar plates and cultured at 25 °C for 14 days under 12 h light/12 h dark conditions (Zou et al. 2010). The holotypes and ex-types cultures were deposited at the Institute of Fungus Resources, Guizhou University (formally Herbarium of Guizhou Agricultural College; code, GZAC), Guiyang City, Guizhou, China. MycoBank numbers were obtained as outlined in MycoBank (http:// www.MycoBank.org) (Crous et al. 2004). Colony characteristics were determined on PDA cultures incubated at 25 °C for 14 days and growth rate, presence of octahedral crystals and colony colors (surface and reverse) were observed. To investigate microscopic characteris- MycoKeys 112: 335-359 (2025), DOI: 10.3897/mycokeys.112.140799 336 Wan-Hao Chen et al.: Four new araneogenous species and a new genus in Hypocreales tics, a little of the mycelia was picked up from the colony and mounted in lac- tophenol cotton blue or 20% lactic acid solution and the asexual morphological characteristics (e.g., conidiophores, phialides or conidiogenous cells, and co- nidia) were observed and measured using a Leica DM4 B microscope. DNA extraction, polymerase chain reaction amplification and nucleotide sequencing DNA extraction was carried out using a fungal genomic DNA extraction kit (DP2033, BioTeke Corporation) according to Liang et al. (2011). The extracted DNA was stored at -20 °C. Polymerase chain reaction (PCR) was used to amplify genetic markers using the following primer pairs: ITS4/ITS5 for the internal tran- scribed spacer (ITS) region (White et al. 1990), LROR/LR5 for 28s large subunit ribosomal (LSU) (Vilgalys and Hester 1990), fRPB2-5F/fRPB2-7cR for RNA poly- merase Il second largest subunit (RPB2) (Liu et al. 1999) and 983F/2218R for translation elongation factor 1 alpha (tef-1a) (Castlebury et al. 2004). The ther- mal cycle of PCR amplification for these phylogenetic markers was set up follow- ing the procedure described by Chen et al. (2021). PCR products were purified and sequenced at Sangon Biotech (Shanghai) Co. All newly generated sequenc- es were deposited in GenBank and accession numbers were obtained (Table 1). Sequence alignments and phylogenetic analyses DNASTAR™ Lasergene (v 6.0) was used to edit DNA sequences in this study. Analyses 1: ITS, LSU and tef-1a sequences for the strains in Clavicipitaceae were downloaded from GenBank based on Xiao et al. (2023), Chen et al. (2024) and other sequences were selected based on BLASTn searches. Analyses 2: ITS, LSU, RPB2 and tef-1a sequences for the strains in Cordycipitaceae were downloaded from GenBank based on Khonsanit et al. (2024) and other se- quences were selected based on BLASTn searches. All the sequences were aligned and edited by MAFFT v.7.037b (Katoh and Standley 2013) and MEGA6 (Tamura et al. 2013). Combined sequences for analysis 1 (dataset 1: ITS, LSU, tef-1a) and analysis 2 (dataset 2: ITS, LSU, RPB2, tef-1a) were obtained us- ing SequenceMatrix v.1.7.8 (Vaidya et al. 2011). The model was selected for Bayesian analysis by ModelFinder (Kalyaanamoorthy et al. 2017) in PhyloSuite (v1.2.2) software (Zhang et al. 2020). The datasets 1 and 2 for analysis 1 and 2 were analyzed using Bayesian in- ference (BI) and maximum likelihood (ML) methods, respectively. For BI, a Mar- kov chain Monte Carlo (MCMC) algorithm was used to generate phylogenetic trees with Bayesian probabilities for the combined sequence datasets using MrBayes v.3.2 (Ronquist et al. 2012). The Bayesian analysis resulted in 20,001 trees after 10,000,000 generations. The first 4,000 trees, representing the burn- in phase of the analysis, were discarded, while the remaining 16,001 trees were used to calculate posterior probabilities in the majority rule consensus tree. Af- ter the analysis was finished, each run was examined if it was greater than 200 using the program Tracer v.1.5 (Drummond and Rambaut 2007) to determine burn-in and confirm that both runs had converged. ML analyses were construct- ed with IQ-TREE (v 2.0) (Trifinopoulos et al. 2016), using an automatic selection of the model according to BIC. MycoKeys 112: 335-359 (2025), DOI: 10.3897/mycokeys.112.140799 337 Wan-Hao Chen et al.: Four new araneogenous species and a new genus in Hypocreales Table 1. List of strains and GenBank accession numbers of sequences used in this study. Species Aciculosporium oplismeni A. take A. take Akanthomyces aculeatus A. aculeatus Aschersonia confluens A. placenta Ascopolyporus albus A. albus Arachnidicola sulphurea A. sulphurea Atkinsonella hypoxylon Balansia epichloe B. henningsiana B. pilulaeformis Beauveria bassiana B. bassiana Blackwellomyces cardinalis B. cardinalis Bhushaniella rubra B. rubra Chlorocillium araneogenum C. araneogenum C. griseum C. griseum C. gueriniae C. gueriniae C. gueriniae C. lepidopterorum C. lepidopterorum C. montefioreae C. guizhouense C. guizhouense C. sinense C. sinense C. vallense C. vallense Claviceps fusiformis C. purpurea C. purpurea Clonostachys rosea Collarina aurantiaca C. aurantiaca Conoideocrella luteorostrata C. luteorostrata C. tenuis Corallocytostroma ornithocopreoides Cordyceps militaris C. militaris C. inthanonensis GenBank Accession No. MAFF 246966. _L0S71760_—-Los7I760. | "MAFF 241224 | —_L0S71753.|—~L0S7I789. «| = “nsr-soass | ucsri7ss | tes7i7s6 | Twuatesus [| Mraies20 7 a sec 7961 | wNoaveai | vaseasa7 | ~~ scc7as9 | JNoavea2 | eracso7a [= [me [DC Oe TAEG.9627a | Noes | AveasTE7 | AcGo«2 [| APSAG7eB TARSEF S64" | waBeo7eT || CH0880905 aRSEF147@ | avssi981 |= ~SS*|SCH0880908 BCC 475417 0Q914433 BCC 47542 0Q914434 KYO7181" KYO7182 ATCC 26019 GAM 12885 FMR 11134 FMR 11784 NHJ 6293 wace7os5 | MN576932 MT017832 MycoKeys 112: 335-359 (2025), DOI: 10.3897/mycokeys.112.140799 tef-1a LC572040 LC572034 LC572035 MF416465 KC519366 DQ384976 EF469056 OL322035 OL322036 MF140843 MF140842 KP689546 EF468743 AY489610 DQ522319 HQ880974 AY531890 EF469059 DQ522325 0Q914428 0Q914429 MW753039 MW753040 MW091327 OR737799 OR737801 OR737802 MW753041 MW753042 PP438400 MN101596 PQ444210 PP766580 PP766581 PQ444211 PQ444212 DQ522320 AF543778 EF469058 AY489611 EF468801 EF468800 EU369029 LT216546 DQ522332 MN576988 338 Wan-Hao Chen et al.: Four new araneogenous species and a new genus in Hypocreales Species GenBank Accession No. teF-ta Corniculantispora psalliotae CBS 532.817 EF469067 C. psalliotae cBs101270 | = - | +EF469081 «| ~S—s«EF469113 | ~—s«EF469066 Corpulentispora magnispora MK336037 Dussiella tuberiformis* ee ee eee ee es eee JQ257027 Engyodontium aranearum DQ522341 Flavocillium acerosum KM283810 Gamszarella buffelskloofina CBS 150062' Helicocollum surathaniensis | pec3446e3 | = -~Ssts~ssSs«éK 202328 «3S KT222336 Heteroepichloe bambusae — Baot | poesa26 H. bambusae a Hevansia novoguineensis MH521885 Hyperdermium bertoni ~arzazasa | CAF2ADSA || Keithomyces carneus CBS 239.32 | NR131993 | NGO57769 | - EF468789 Lecanicillium lecanii DQ522359 Marquandomyces marquandii CBS 182.27, | ~NR131994 | EFA68845 | = EF468793 Metapochonia bulbillosa | CBS145.70 | MH859529 | AF339542, | EF468796 M.gonioides ~ceseo72 | Alzezaoo —|—AFSS9550. = ~=SCSC«éS22854 MycoKeys 112: 335-359 (2025), DOI: 10.3897/mycokeys.112.140799 339 Wan-Hao Chen et al.: Four new araneogenous species and a new genus in Hypocreales Species Metarhiziopsis microspora M. microspora Metarhizium anisopliae M. anisopliae M. flavoviride M. flavoviride M. flavoviride Microhilum oncoperae Moelleriella phyllogena M. phyllogena M. umbospora Morakotia fusca M. fusca Mycophilomyces periconiae Myriogenospora atramentosa Neoaraneomyces araneicola N. araneicola Neobaryopsis andensis N. andensis Neobarya parasitica Neogamszarella antillata Neohyperdermium piperis N. pulvinatum Neotorrubiella chinghridicola N. chinghridicola Niesslia exilis Nigelia aurantiaca N. martiale Niveomyces coronatus N. coronatus Orbiocrella petchii O. petchii Papiliomyces liangshanensis P liangshanensis P. shibinensis Parahevansia koratensis P websteri Parametarhizium changbaiense P. hingganense Paraneoaraneomyces sinensis P. sinensis Parengyodontium album P album Parepichloe cinerea Periglandula ipomoeae Pleurodesmospora coccorum P lepidopterorum P lepidopterorum Pochonia boninensis P. chlamydosporia Polystromomyces araneae GenBank Accession No. "ceHSTa3a | —eFaease9 —|~—erasasm@ “INeHS1330—|—EFaease9 | =EFAGAS72. arses? | Hoss oes 1307"" | wrovesea | MTO7e8ss | “cas 12565 | MTo7eees —wroveesa | cas70074 |; MToveess | ~ops218.56" | MHBS7590—wHe6sT99 | —aRser 4958-9952. —|—_EFAGODI6 oupos7es | | ussae10 | ~ouros7s3 | | SCussze08 | ouros7ei” [=| ussze8 | ~sccea2s | KY ISB | pocrearay [= KY ISABG1 | orc a7sss | NRIS#209 | NG.OSS746 | acceca2 |: | AaB] | priori" | Mw7a0520 | ww7a0609 | ~pvioi712_ | mw7a0s22 | Mw7a0600 | AF 259672" | NRI69713 | NG.O68701 | ~arasse7-1 | Mrisg956_ | MT1s9005. | arson sin_|_KP899626 | KPS09626. eesniemiay [=| AvA66442 | FU369089 po.602 | = ~| =~ barre73e | Arzazas3 ~8ccas6e4 | wKe32098 | MK692090. rT eessso7m [| AvABO720. | pecans | | USTOMe rT erccesss [= | Cranso7a | ~wvoassasoo" |= | ~ON49606 | _ONST3A00 Neo [= | ~ONAD60S | ~NHJ6209 | uNoasasT | eus60099 | fe Cerocvasz [| ~Craseets | erectszs | ~~S=~Caseota | GZUH NR154178 SB13050311' oe ae a ~cemcc 19143" | MNS89741 | MNSB9994. | “cGMcc 19144 | MNOSS703.———MNOGI635. | ~—zvz2006 | 0709254 og708260 ~~ zv22007 | 0709255 og7os2e7 | ~ease96.71 | Lcos2ee2 | MMevaie | "cas 368.72 | MHe60s02 | MHe7217 | Neo! | ABoes25 | el eas 460.73 | wigeo7as Heras | ~pytosor | wwezes76 |; | MNERABTG "py i0s02 | Mwezes77 | SSNS a3 18 ~~ yew19597 | Abvosese | AB7O0ET | "ees 101244 | JNoaseat | asie7s8 | MycoKeys 112: 335-359 (2025), DOI: 10.3897/mycokeys.112.140799 tef-1a DQ463996 MT078845 MT078846 MT078847 KJ398787 EF468785 EU392674 EU392672 EU392688 KY794857 KY794856 AY489628 MW753033 MW753034 DQ522350 DQ118749 DQ118746 MK632072 AY489614 GU979957 JF416016 ON513397 EU369023 EU369022 EF468756 EF468755 KR153589 GQ250031 GQ250032 MN908589 MN065770 0Q719626 0Q719627 LC382178 LC382183 KP689568 MW834317 MW834319 AB758463 DQ522327 MZ707825 340 Wan-Hao Chen et al.: Four new araneogenous species and a new genus in Hypocreales Species Pseudogibellula formicarum P. formicarum Pseudoniveomyces arachnovorum P blattae P blattae Pseudolecanicillium caatingaense P caatingaense Purpureomyces maesotensis P. maesotensis P. maesotensis Regiocrella camerunensis Rotiferophthora angustispora Samsoniella alboaurantium S. alboaurantium S. inthanonensis S. inthanonensis Samuelsia chalalensis S. mundiveteris S. rufobrunnea Shimizuomyces paradoxus S. paradoxus Simplicillium lanosoniveum Sungia yongmunensis S. yongmunensis Tyrannicordyceps fratricida Ustilaginoidea dichromonae U. virens U. virens Yosiokobayasia kusanagiensis Zarea fungicola Zouia cauligalbarum Z. cauligalbarum Pleurocordyceps aurantiaca P. marginaliradians Purpureocillium lilacinum Strain GenBank Accession No. ITS LSU RPB2 tef-1a BCC 84257 MT508782 MT512653 - MT533480 CBS:433.73 MH860731 MH872442 - MT533481 BCC 95818" ORO98526 - - OR133173 BGG 53567" ON103042 ON103167 ON125036 - BCC 53568 ON103043 ON103168 ON125037 ON125025 URM8446' ON862933 ON862925 OP290514 OP290526 URM8442 ON862934 ON862926 OP290513 OP290525 BCC 88441 MN781916 MN781877 - MN781734 BCC 85349 MN781928 MN781872 - MN781729 BCC 89300' MN781917 MN781876 - MN781733 ARSEF 7682 - DQ118735 - DQ118743 CBS 101437 AJ292412 AF339535 - AF543776 CBS 262.58' AY624179 AB080087 MF416448 MF416497 CBS 240.32 AY624178 JF415979 JF415999 JF416019 TBRC 7915" NR_164420 NG_069500 MF140815 MF140849 TBRC 7916 MF140760 MF140724 MF140814 MF140848 CUP 067856" - EU392637 - EU392691 BCC 40021 - GU552152 - GU552145 CUP 067858 - AY986918 - AY986944 EFCC 6279 JN049847 EF469084 - EF469071 EFCC 6564 - EF469083 - EF469072 CBS 123.42 NR_171734 NG_068571 - - EFCC 2131 JN049856 EF468833 - EF468770 EFCC2135 - EF468834 - EF468769 TNS-F 19011 JQ349068 JQ257023 - JQ257028 MRL IB9228 - ; - JQ257025 ATCC 16180 - - - JQ257026 MAFF 240421 - JQ257011 - JQ257024 TNS-F18494 - JEAI59 72 - JF416014 CBS 992.69' NR_119653 KM283792 KM283857 KM283816 GZUIFRZHJ01" MH730663 MH730667 MH801924 MH801920 GZUIFRZHJ02 MH730664 MH730668 MH801925 MH801921 MFLUCC 17-2113 MG136916 MG136910 - MG136875 MEFLU 17-1582" MG136920 MG136914 - MG136878 CBS 431.87 AY624188 EF468844 EF468940 EF468791 Note: New strains or species are in bold type. * J.F. White, Scale on Arundinaria tecta, North Carolina, 2000. “"” denotes ex-type. Abbre- viations: ARSEF, USDA-ARS Collection of Entomopathogenic Fungal cultures, Ithaca, NY; ATCC, American Type Culture Collection, USA; BCC, BIOTEC Culture Collection, KlongLuang, Thailand; CBS, Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands; CGMCC, China General Microbiological Culture Collection Center, China; EFCC, Entomopathogenic Fungal Culture Collection, Chuncheon, Korea; GZUH, Guizhou University Herbarium, Guiyang, Guizhou, China; HMAS, Herbarium of Mycology, Chinese Academy of Sciences; MAFF, Ministry of Agriculture, Forestry and Fisheries of Japan, Tokyo, Japan; NHJ, Nigel Hywel-Jones personal collection; OSC, Oregon State University Herbarium, Corvallis, OR; TNS-F, the mycological herbarium of the National Museum of Nature and Science, Tsukuba, Ibaraki, Japan; WAC, Western Australian Plant Pathology Reference Culture Collection, Australia, Perth. Genealogical Concordance Phylogenetic Species Recognition (GCPSR) analysis The Genealogical Concordance Phylogenetic Species Recognition model was applied to analysis the related species. The pairwise homoplasy index (PHI) (Bruen et al. 2006) is a model test based on the fact that multiple gene phy- logenies will be concordant between species and discordant due to recombi- nation and mutations within a species. The test was performed in Splits Tree4 (Huson and Bryant 2006) as described by Quaedvlieg et al. (2014) to determine MycoKeys 112: 335-359 (2025), DOI: 10.3897/mycokeys.112.140799 34] Wan-Hao Chen et al.: Four new araneogenous species and a new genus in Hypocreales the recombination level within phylogenetically closely-related species using a three-locus or four-locus concatenated dataset. The new species and their closely-related species were analyzed using this model. The relationships be- tween closely-related species were visualized by constructing a split graph, us- ing both the LogDet transformation and splits decomposition options. Results Phylogenetic analyses Analysis 1: Phylogenetic trees were generated in analysis 1 to determine the establishment of the new Chlorocillium species in Clavicipitaceae (Fig. 1). Pleu- rocordyceps aurantiaca (Y.P. Xiao et al.) Y.H. Wang et al. (MFLUCC 17-2113) and P. marginaliradians (Y.P. Xiao et al.) Y.H. Wang et al. (MFLU 17-1582) were used as the outgroup taxa in the analysis. The dataset included 74 taxa, and consisted of 2,280 (ITS, 660; LSU, 740, and tef-1a, 880) characters with gaps. The selected model for ML analysis was TIM+F+l+G4. The final value of the highest scoring tree was —26,825.060, which was obtained from the ML analy- sis of the dataset. The parameters of the GTR model used to analyze the data- set were estimated based on the following frequencies: A = 0.224, C = 0.291, G = 0.279, T = 0.207; substitution rates AC = 1.00000, AG = 2.11049, AT = 1.16800, CG = 1.16800, CT = 5.30232 and GT = 1.00000, as well as the gamma distribution shape parameter a = 0.474. The selected model of the dataset for BI analysis was GTR+tF+l+G4. The phylogenetic tree (Fig. 1) constructed using ML and BI analyses was largely congruent and strongly supported in most branches. Most genera clustered into independent clades. Strains DL10171, DL10172, DY09021 and DY09022 clustered into two independent clades. Strains DL10171 and DL10172 have a close relationship with Chlorocillium griseum, whereas strains DY09021 and DY09022 have a close relationship with C. sinense W.H. Chen et al. and C. araneogenum (W.H. Chen et al.) W.H. Chen, et al. Analysis 2: The phylogenetic trees were generated in analysis 2 to determine the establishment of the new species in Cordycipitaceae (Fig. 2). Purpureocilli- um lilacinum (Thom) Luangsa-ard et al. (CBS 431.87) was used as the outgroup taxon in the analysis. The dataset included 49 taxa and consisted of 2,280 (ITS, 631; LSU, 782, RPB2, 833 and tef-1a, 893) characters with gaps. The selected model for ML analysis was TIM3+F+l+G4. The final value of the highest scoring tree was —36,309.393, which was obtained from the ML analy- sis of the dataset. The parameters of the GTR model used to analyze the data- set were estimated based on the following frequencies: A = 0.237, C = 0.280, G = 0.272, T = 0.211; substitution rates AC = 1.16611, AG = 2.87992, AT = 1.00000, CG = 1.16611, CT = 6.74648 and GT = 1.00000, as well as the gamma distribution shape parameter a = 0.445. The selected model for BI analysis was GTR+F+l+G4 (ITS), GTR+F+Il+G4 (LSU and tef-1a) and SYM+G4 (RPB2). The phylogenetic trees (Fig. 2) constructed using ML and BI analyses were largely congruent and strong- ly supported in most branches. Most genera clustered into independent clades. Strains WD04081, WD04082, WD04101 and WD04102 clustered into two indepen- dent clades with high statistical support (100% ML/1 PP) and were clustered with Gamszarella antillana (R.F. Castafeda & G.R.W. Arnold) Crous and G. buffelskloofina Crous ina clade with high statistical support in ML and BI analysis (100% ML/1 PP). MycoKeys 112: 335-359 (2025), DOI: 10.3897/mycokeys.112.140799 349 Wan-Hao Chen et al.: Four new araneogenous species and a new genus in Hypocreales Myriogenospora atramentosa A.E.G 96-32 100/1 ~~ Balansia henningsiana A.E.G. 96-27a Balansia epichloe A.E.G. 96-15a 94/0.69 Balansia pilulaeformis A.E.G. 94-2 100/1'— A tkinsonella hypoxylon B4728 Periglandula ipomoeae lasaF13 100/1| Morakotia fusca BCC 792727 soj-| 86/1 Morakotia fusca BCC 64125 87/4 Regiocrella camerunensis ARSEF 7682 87/1 Shimizuomyces paradoxus EFCC 6564 55/- nue Shimizuomyces paradoxus EFCC 6279 Heteroepichloe sasae E.sasae-N we Heteroepichloe sasae E.sasae-H Heteroepichloe bambusae Bo-01 Heteroepichloe bambusae Ba-01 100/1 Aschersonia confluens BCC 7961 Aschersonia placenta BCC 7869 9910.83 Moelleriella umbospora CUP 067817 99/0.89 Moelleriella phyllogena CUP 067793 100/0.98 = Moelleriella phyllogena CUP 067785 Corallocytostroma ornithocopreoides WAC 8705 185/0.65 Aciculosporium oplismeni MAFF 246966 100/1|_| Aciculosporium take TNS-F-60465 navi Aciculosporium take MAFF 241224 64/- Neobarya parasitica Marson s/n Samuelsia mundiveteris BCC 40021 98/0.68 Samuelsia rufobrunnea CUP 067858 9910.96 Samuelsia chalalensis CUP 0678567 Dussiella tuberiformis 50/- Claviceps fusiformis ATCC 26019 7510.61 Claviceps purpurea S.A. cp11 100/1 Claviceps purpurea GAM 12885 99/0.90 Purpureomyces maesotensis BCC 85349 Purpureomyces maesotensis BCC 893007 Purpureomyces maesotensis BCC 88441 Avel| OU Yosiokobayasia kusanagiensis TNS-F 18494 100/17 Metarhizium anisopliae CBS 130.71 7 Metarhizium anisopliae ARSEF 7487 100/1 Metarhizium flavoviride CBS 700.74 100] Metarhizium flavoviride CBS 218.56 7 Metarhizium flavoviride CBS 125.65 Mycophilomyces periconiae CPC 27558 94/0.64 Collarina aurantiaca FMR 11784 100/1" Collarina aurantiaca FMR 11134 100/1 | Paraneoaraneomyces sinensis ZY 22.007 100/1 92/0.89 100/1 100/1 51/- Paraneoaraneomyces sinensis ZY 22.006 ety Neoaraneomyces araneicola DY101712 eval Neoaraneomycesaraneicola DY101711* 100/1 | Sungia yongmunensis EFCC 2135 Sungia yongmunensis EFCC 2131 100/1 F Epichloe elymi C. Schardl 760 64/- Epichloe typhina ATCC 56429 100/1 | Conoideocrella tenuis NHJ 6293 Conoideocrella luteorostrata NHJ 12516 Conoideocrella luteorostrata NHJ 11343 Tyrannicordyceps fratricida TNS-F 19011 81/-L_| Helicocollum surathaniensis BCC 344647 100/1" Helicocollum surathaniensis BCC 34463 93/0.59 Keithomyces carneus CBS 239.32 Marquandomyces marquandii CBS 182.27 98/0.95 Rotiferophthora angustispora CBS 101437 bo/0.56 Parametarhizium changbaiense CGMCC 19144 100/1 " Parametarhizium changbaiense CGMCC 191437 100/1f Metapochonia rubescens CBS 464.88 7 Metapochonia sulchlasporia CBS 251.83 98/1 Metapochonia gonioides CBS 891.72 100/1" Metapochonia bulbillosa CBS 145.70 100/1- Pochonia boninensis JCM 18597 Pochonia chlamydosporia CBS 101244 86/0.73 Papiliomyces shibinensis GZUH $B13050311 7 98/0.89 Papiliomyces liangshanensis EFCC 1523 100/1" Papiliomyces liangshanensis EFCC 1452 99/17 Orbiocrella petchii NHJ 6240 65/- Orbiocrella petchii NHJ 6209 Clonostachys rosea GJS90-227 100/0.67'— Niesslia exilis CBS 560.74 Parepichloe cinerea Ne-01 51/- Ephelis tripsaci_ CBS 857.72 7 100/0.99|_| Ephelis japonica Eph.oryzae ““ Ephelis japonica CBS 236.64 Ustilaginoidea dichromonae MRL IB9228 90/0.94 Ustilaginoidea virens MAFF 240421 ool 00/1 Ustilaginoidea virens ATCC 16180 Nigelia martiale EFCC 6863 100/!- Nigelia aurantiaca BCC 13019 Chlorocillium gueriniae BRIP 72668a 100/1) Chlorocillium gueriniae BRIP 72680a? 100/1 Chlorocillium gueriniae BRIP 72666a Chlorocillium montefioreae BRIP 70299a™ 56/- 100/1 50/- 64/0.58 ay 100/1 | Chlorocillium lepidopterorum SD05362 100/0.77 Chlorocillium lepidopterorum SD05361 91 _| Chlorocillium griseum RCEF6632 FO0/ Chlorocillium griseum CBS 387.73 7 100/1| Chlorocillium guizhouense DL10172 Chlorocillium guizhouense DL101717 Chlorocillium sinense KY07182 93/0.57 aa : ahage 4d Chlorocillium sinense KY07181 7 7970.53 400/922 Chlorocillium araneogenum DY101802 Chlorocillium araneogenum DY 1018017 Chlorocillium vallense DY09022 9910.99 " Chlorocillium vallense DY090217 Metarhiziopsis microspore INEHS133a 100/1" Metarhiziopsis microspore CEHS133a Pleurocordyceps aurantiaca MFLUCC 17-2113 Pleurocordyceps marginaliradians MFLU 17-1582 100/0.98 100/1 0.05 Figure 1. Phylogram retrieved from IQTREE of the new species and related species in Clavicipitaceae using the combined dataset of ITS, LSU, and tef-1a gene regions. The statistical values are provided at nodes as ML/PP (ML value above 50% and BI value above 0.50). The tree is rooted with Pleurocordyceps aurantiaca (MFLUCC 17-2113) and P. marginaliradians (MFLU 17-1582). Ex-types, new strains and new species are indicated by the superscript “T” and in bold, respectively. MycoKeys 112: 335-359 (2025), DOI: 10.3897/mycokeys.112.140799 343 Wan-Hao Chen et al.: Four new araneogenous species and a new genus in Hypocreales 100/1) Arachnidicola sulphurea TBRC 7249 68/- Arachnidicola sulphurea TBRC 72487 Akanthomyces aculeatus TS772 100/1' Akanthomyces aculeatus HUA 1861457 94/0.65 Lecanicillium lecanii CBS 102067 * 100/1! Tecanicillium lecanii CBS 101247 Kanoksria zaquensis HMAS 246917 88/0.601 100/1' Kanoksria zaquensis HMAS 2469157 100/1 | Samsoniella inthanonensis TBRC 7916 Samsoniella inthanonensis TBRC 7915 100/1) 1 Samsoniella alboaurantium CBS 262.58 * 100/11 co msoniella alboaurantium CBS 240.32 Parahevansia websteri NHJ 2662 100/1! Parahevansia koratensis BCC 01485 1h0/0.98| Beauveria bassiana ARSEF 1564 7 Beauveria bassiana ARSEF 1478 op /0.90 Microhilum oncoperae ARSEF 4358 100/17 Cordyceps militaris YFCC 6587 Cordyceps militaris OSC 936237 98/0.99 Cordyceps inthanonensis BCC 56302 anal Cordycepsinthanonensis BCC 558127 Pleurodesmospora coccorum CBS 460.73 95/0.77 96/0.60 96/0.60] 00/1 100/1 Pleurodesmospora lepidopterorum DY10502 SHE 195/0.99 gal Pleurodesmospora lepidopterorum DY105017 Neohyperdermium piperis CBS 116719 7 100/0.75 Neohyperdermium pulvinatumP.C. 602 98/- Corniculantispora dimorpha CBS 363.86 * 9910.68 Corniculantispora psalliotae CBS 532.81 7 ie 96/0.89 Corniculantispora aranearum CBS 797.84 * 100/1" Corniculantispora dimorpha CBS 345.37 Corniculantispora psalliotae CBS 101270 60 Liangia sinensis YFCC 3104 100/1' Liangia sinensis YFCC 31037 61/-| _ Hyperdermium bertonii AF242354 Bhushaniella rubra BCC 47542 100/ Bhushaniella rubra BCC 475417 Pseudolecanicillium caatingaense URM8446" 100/1" Pseudolecanicillium caatingaense URM8442 97/1 Zarea fungicola CBS 992.69 7 87/0.98 Engyodontium aranearum CBS 309.85 62/- 96/0.80 Ascopolyporus albus BCC 48976 100/!' Ascopolyporus albus BCC 489757 100/1] Blackwellomyces cardinalis OSC 93610 5/0.82 Blackwellomyces cardinalis OSC 936097 Zouia cauligalbarum GZUIFRZHJ02 100/1' Zouia cauligalbarum GZUIFRZHJ01 * 10-50) toon Niveomyces coronatus Niveo Niveomyces coronatus NY04434800 7 gu Pseudogibellulaformicarum CBS 433.73 too Pseudogibellula formicarum BCC 84257 100/17— Flavocillium bifurcatum YFCC 61017 Flavocillium acerosum CBS 418.81 7 100/1 | Gibellula nigeli NHJ 108087 Gibellula nigeli BCC 47555 68/0.51 Polystromomyces araneae BCC 933017 Pseudoniveomyces blattae BCC 53568 96/0.80 100/1' Pseudoniveomyces blattae BCC 535677 63/- | 100/17 Hevansia novoguineensis CBS 610.80 7 Hevansia novoguineensis BCC 47881 7510.96 Jenniferia cinerea BCC 2191 100/1" Jenniferia cinerea BCC 06839 100/17 Neotorrubiella chinghridicola BCC 807337 Neotorrubiella chinghridicola BCC 39684 gon | 1004 Corpulentispora magnispora LC12469 Corpulentispora magnispora CGMCC 3.193047 89/0.89 Neogamszarella antillata CBS 350.857 62/- Naa Gamszarella buffelskloofina CBS 150062 7 sana 100/1| Gamszarella vallensis WD04102 Gamszarella vallensis WD041017 100/1] | Gamszarella sinensis WD04082 100/1" Gamszarella sinensis WD040817 100/1 Parengyodontium album CBS 836.71 Parengyodontium album CBS 368.72 80/0.75 10/1 Gamszarea humicola CGMCC 3.19303 * 90/0.88 Gamszarea wallacei CBS 101237 7 100/1f Leptobacillium chinense CGMCC 3.149707 Leptobacillium leptobactrum CBS 774.69 7 100/1 Leptobacillium filiforme URM 79187 93/0.84|__| Leptobacillium coffeanum COAD 2061 100/1' Leptobacillium coffeanum COAD 20577 Simplicillium lanosoniveum CBS 123.42 7 Neobaryopsis andensis A.F.25967-27 100/1' Neobaryopsis andensis A.F.25967-1 Purpureocillium lilacinum CBS 431.87 Figure 2. Phylogram retrieved from IQTREE of the new species and other related species in Cordycipitaceae using the combined dataset of ITS, LSU, RPB2 and tef-1a gene regions. The statistical values are provided at nodes as ML/PP (ML value above 50% and BI value above 0.50). The tree is rooted with Purpureocillium lilacinum (CBS 431.87). Ex-types, new strains and new species are indicated by the superscript “T” and in bold, respectively. MycoKeys 112: 335-359 (2025), DOI: 10.3897/mycokeys.112.140799 344 Wan-Hao Chen et al.: Four new araneogenous species and a new genus in Hypocreales Genealogical Concordance Phylogenetic Species Recognition (GCPSR) analysis A three-locus concatenated dataset (ITS, LSU and tef-1a) was used to deter- mine the recombination level within Chlorocillium araneogenum (DY101801), C. griseum (CBS 387.73), C. gueriniae (BRIP 72680a), C. montefioreae (BRI- P70299a), C. lepidopterorum (SD05361), C. guizhouense (DL10171), and strains KY07181 and DY09021 (Fig. 3), whereas a four-locus concatenated dataset (ITS, LSU, RPB2 and tef-1a) was used to determine the recombination level within Gamszarella antillana (CBS 350.85), G. buffelskloofina (CBS 150062), and strains WD04081 and WD04101 (Fig. 4). Chaiwan et al. (2022) noted that if a | 0.001 Chlorocillium lepidopterorum Chlorocillium gueriniae eat BRIP 72680a Chlorocillium araneogenum DY101801 h - Cate Chlorocillium 7% montefioreae BRIP70299a Chlorocillium griseum DL10171 <————- — — CBS 387.73 UY Chlorocillium DY09021 sinense KY07181 Figure 3. Results of the pairwise homoplasy index (PHI) test of the new Chlorocillium strains and its closely-related species using both LogDet transformation and splits decomposition. PHI test results (®,) < 0.05 indicate significant recombination within the dataset. The new strains are in bold type. Gamszarella antillata CBS 350.85 A -——10.01 Gamszarella buffelskloofina > CBS 150062 WD04101 < v WD04081 Figure 4. Results of the pairwise homoplasy index (PHI) test of the new Gamszarella strains and its closely-related spe- cies using both LogDet transformation and splits decomposition. PHI test results (®, ) < 0.05 indicate significant recom- bination within the dataset. The new strains are in bold type. MycoKeys 112: 335-359 (2025), DOI: 10.3897/mycokeys.112.140799 345 Wan-Hao Chen et al.: Four new araneogenous species and a new genus in Hypocreales the PHI is below the 0.05 threshold (®, < 0.05), it indicates that there is signif- icant recombination in the dataset. This means that related species in a group and recombination levels are not different. If the PHI is above the 0.05 thresh- old (®, > 0.05), it indicates that it is not significant, which means that the relat- ed species in a group level are different. The result of the pairwise homoplasy index (PHI) test of Chlorocillium araneogenum, C. griseum, C. gueriniae, C. mon- tefioreae, C. lepidopterorum, C. guizhouense, strains KY07181 and DY09021 was 1.0 and revealed that those species and strains KY07181 and DY09021 were different (Fig. 3). The result of the pairwise homoplasy index (PHI) test of Gamszarella antillana, G. buffelskloofina, and strains WD04081 and WD04101 was 1.0 and revealed that those species and strains WD04081 and WD04101 were different (Fig. 4). Taxonomy Chlorocillium Zare & W. Gams, Mycol. Progr. 15: 1005, 2016 Chlorocillium guizhouense W.H. Chen, Y.F. Han & J.D. Liang, sp. nov. MycoBank No: 856175 Fig. 5 Etymology. Referring to the location, Guizhou Province, where the type speci- men was collected. Type. CHINA * Guizhou Province, Qiandongnan Miao and Dong Auton- omous Prefecture, Rongjiang County, Dali Dong Village (26°01'58.70'N, 108°24'48.06"E). On a dead spider (Araneae), 1 October 2018, Wanhao Chen, GZAC DL1017 (holotype), ex-type, DL10171. Description. Colonies on PDA reaching 21-24 mm diam. in 14 d at 25 °C, white, consisting of a basal felt and cottony, floccose overgrowth, reverse yel- lowish. Prostrate hyphae smooth, septate, hyaline, 1.1-1.6 um diam. Conidi- al structures consisting of erect conidiophores usually arising from the aerial hyphae, solitary or lecanicillium-like with phialides in whorls of two to three. Phialides 11.6-25.3 x 1.0-1.2 um, with a cylindrical basal portion, tapering into a distinct neck. Conidia in chains, hyaline, fusiform, 1-celled, 2.6-3.8 x 1.1- 1.6 um (average values 3.2 x 1.3 um). Octahedral crystals not observed. Host. Spider (Araneae). Additional material examined. CHINA * Guizhou, Qiandongnan Miao and Dong Autonomous Prefecture, Rongjiang County, Dali Dong Village (26°01'58.70'N, 108°24'48.06"E). On a dead spider (Araneae), 1 October 2018, Wanhao Chen, DL10172 (living culture). Remarks. Chlorocillium guizhouense was identified as Chlorocillium, based on the BLASTn result in NCBI and the phylogenetic analysis of the combined dataset 1 (Fig. 1). It clustered into an independent clade with a close rela- tionship with C. araneogenum, C. sinense and C. vallense with high bootstrap value (93% ML). Compared with the typical characteristics of the known spe- cies (Table 2), C. guizhouense was distinguished from C. araneogenum by its smaller conidia (2.6—-3.8 x 1.1-1.6 um vs. 3.4—5.8 x 1.4-1.8 um). Chlorocillium guizhouense can be distinguished from C. sinense by its larger conidia (fusi- form, 2.6-3.8 x 1.1-1.6 um vs. fusiform to ellipsoidal, 1.9-2.9 x 0.8-1.2 um). MycoKeys 112: 335-359 (2025), DOI: 10.3897/mycokeys.112.140799 346 Wan-Hao Chen et al.: Four new araneogenous species and a new genus in Hypocreales C Figure 5. Chlorocillium guizhouense A infected spider B, C PDA culture plate showing top (B) and reverse (C) sides of the colony D-F phialides and conidia were stained with cotton blue G—J phialides and conidia. Scale bars: 10 mm (B, C); 10 um (D-J). Table 2. Morphological comparison of the new species with other Chlorocillium species. C. sinense 11.7-20.1 ¥1..1-1.3 fusiform to ellipsoidal, spider Absent Chen et al. 2024 1.9-2.9 x 0.8-1.2 C.guizhouense | 11.6-25.3%1.0-1.2 fusiform, 2.6-3.8*1.1-1.6 spider = Absent =——_——‘This study C. vallense 9.2-17.1x0.8-1.2 | fusiform, 2.0-3.2x0.9-1.4 spider = Absent =——_—_—‘This study Chlorocillium guizhouense was distinguished from C. vallense by its larg- er phialides (11.6-25.3 x 1.0-1.2 um vs. 9.2-17.1 x 0.8-1.2 um). Thus, the morphological characteristics and molecular phylogenetic results support C. guizhouense as a new species. Mycokeys 112: 335-359 (2025), DOI: 10.3897/mycokeys.112.140799 347 Wan-Hao Chen et al.: Four new araneogenous species and a new genus in Hypocreales Chlorocillium vallense W.H. Chen, Y.F. Han & J.D. Liang, sp. nov. MycoBank No: 856176 Fig. 6 Etymology. Referring to its location, Mayao River Valley, where the fungus was first discovered. Type. CHINA * Guizhou Province, Qiannan Buyi and Miao Autonomous Pre- fecture, Duyun City, Mayao River Valley (26°21'24.71"N, 107°22'48.22"E). Ona dead spider (Araneae), 4 September 2021, Wanhao Chen, GZAC DY0902 (holo- type), ex-type, DY09021. Description. Colonies on PDA reaching 72-74 mm diam. in 14 days at 25 °C, white, consisting of a basal felt and cottony, floccose overgrowth, reverse yel- lowish. Prostrate hyphae smooth, septate, hyaline, 0.9-1.3 um diam. Conidial structures consisting of erect branching conidiophores usually arising from the aerial hyphae, solitary or lecanicillium-like in whorls of two to four. Phialides 9.2-17.1 x 0.8-1.2 um, with a cylindrical basal portion, tapering into a distinct neck. Conidia hyaline, fusiform, 1-celled, 2.0-3.2 x 0.9-1.4 um (average values 2.4 x 1.2 um). Octahedral crystals not observed. Host. Spider (Araneae). Additional strain examined. CHINA + Guizhou Province, Qiannan Buyi and Miao Autonomous Prefecture, Duyun City, Mayao River Valley (26°21'24.71'N, 107°22'48.22"E). On a dead spider (Araneae), 4 September 2021, Wanhao Chen, DY09022 (living culture). Remarks. Chlorocillium vallense was identified as in Chlorocillium s. str., based on the BLASTn result in NCBI and the phylogenetic analysis of the aeeme or ‘ rf i= — ” | ' ’ a! a a é' 4E “Ne oe > TJ “ Figure 6. Chlorocillium vallense A infected spider B, C PDA culture plate showing top (B) and reverse (C) sides of the colony D-J phialides and conidia. Scale bars: 10 mm (B, C); 10 um (D-J). MycoKeys 112: 335-359 (2025), DOI: 10.3897/mycokeys.112.140799 348 Wan-Hao Chen et al.: Four new araneogenous species and a new genus in Hypocreales combined dataset 1 (Fig. 1) and clustered into an independent clade with a close relationship with C. sinense and C. araneogenum. Compared with the typical characteristics of the known species (Table 2), C. vallense was dis- tinguished from C. sinense by its shorter phialide (9.2-17.1 x 0.8-1.2 um vs. 11.7-20.1 x 1.1-1.3 um), larger fusiform conidia (2.0-3.2 x 0.9-1.4 um vs. 1.9-2.9 x 0.8-1.2 um) and fast-growing in PDA. Chlorocillium vallense was dis- tinguished from C. araneogenum by its shorter phialide (9.2-17.1 x 0.8-1.2 um vs. 8.3-23.3 x 1.3-2.2 um) and smaller conidia (2.0-3.2 x 0.9-1.4 um vs. 3.4—5.8 x 1.4-1.8 um). Thus, based on both morphological characteristics and molecular phylogenetic results, we confirm C. vallense as a new species. Gamszarella Crous, Persoonia 51: 391, 2023 Gamszarella sinensis W.H. Chen, Y.F. Han & J.D. Liang, sp. nov. MycoBank No: 856388 Figur: Etymology. Referring to the country, China, where the fungus was first discovered. Type. CHINA * Guizhou Province, Guiyang City, Wudang District, Bala Valley (26°45'7.0344"N, 106°58'57.09"E). On a dead spider (Araneae), 5 April 2024, Wanhao Chen, GZAC WD0408 (holotype), ex-type, WD04081. Description. Colonies on PDA reaching 37-38 mm diam in 14 d at 25 °C, white, consisting of a basal felt and cottony, floccose overgrowth, reverse yellowish, with radial patterns. Prostrate hyphae smooth, septate, hyaline, 1.5-1.9 um diam. Co- nidial structures consisting of erect conidiophores usually arising from the aerial hyphae, solitary or lecanicillium-like with conidiogenous cells in whorls of two to three. Conidiogenous cells 7.0-12.0 x 1.0-1.5 um, with a cylindrical basal portion, bearing numerous denticles, tapering into a distinct neck. Conidia hyaline, ellipsoi- dal to fusiform, 1-celled, 2.4-3.9 x 1.5-2.8 um. Octahedral crystals not observed. Host. Spider (Araneae). Additional material examined. CHINA * Guizhou Province, Guiyang City, Wu- dang District, Bala Valley (26°45'7.0344"N, 106°58'57.09"E). On a dead spider (Araneae), 5 April 2024, Wanhao Chen, WD04082 (living culture). Remarks. The new strains were identified as a member in Gamszarella, based on the BLASTn result in NCBI. The phylogenetic analyses of the com- bined dataset 2 (Fig. 2) showed that the new strains clustered as an indepen- dent clade with a close relationship to Gamszarella antillana (R.F. Castafieda & G.R.W. Arnold) Crous and G. buffelskloofina Crous. Compared with the typi- cal characteristics of the known species (Table 3), the new taxon, Gamszarel- la sinensis was distinguished from G. antillana by its shorter conidiogenous cells bearing numerous denticles (Numerous denticles, 7.0-12.0 x 1.0-1.5 um vs. absent of denticles, 18-31 x 1 ym), ellipsoidal to fusiform conidia and absent of octahedral crystals. Gamszarella sinensis was distinguished from G. buffelskloofina by its shorter conidiogenous cells (7.0-12.0 x 1.0-1.5 um vs. 7-22 x 1.5-2 um), smaller ellipsoidal to fusiform conidia [2.4-3.9 x 1.5- 2.8 um vs. (3-)4-6(-10) x 2 um] and spider host. Gamszarella sinensis was distinguished from G. vallensis by its longer conidiogenous cells (7.0-12.0 x 1.0-1.5 um vs. 3.8-5.4 x 1.3-1.9 um). Thus, the morphological characteristics and molecular phylogenetic results support G. sinensis as a new species. MycoKeys 112: 335-359 (2025), DOI: 10.3897/mycokeys.112.140799 349 Wan-Hao Chen et al.: Four new araneogenous species and a new genus in Hypocreales Figure 7. Gamszarella sinensis A infected spider (Araneae) B, C PDA culture plate showing top (B) and reverse (C) sides of the colony D-L conidiogenous cells and conidia were stained with cotton blue M conidiogenous cells and conidia. Scale bars: 10 mm (B, C); 10 um (D-M). Table 3. Morphological comparison of the new species with other Gamszarella species. ; ia ae Octahedral Hosts/ Species Conidiogenous cells (uum) Conidia (um) References G. antillana No denticles, 18-31 x 1 Two types: primary conidia fusiform, Present Agaric Zare and 11-18 x 0.8-1.5; secondary conidia Gams 2001 ellipsoidal, 3-4 x 0.8-1.2 G. buffelskloofina | Terminating in a cluster Subcylindrical, (3-)4—6(-10) x 2 Absent Insect Crous et al. of denticles, 7-22 x 1.5-2 2023 G. sinensis Numerous denticles, Ellipsoidal to fusiform, This study 7.0-12.0 x 1.0-1.5 2.4-3.9 x 1.5-2.8 G. vallensis Numerous denticles, Ellipsoidal to fusiform, This study 3.8-5.4 x 1.3-1.9 2.3-3.0 x 1.7-1.9 Gamszarella vallensis W.H. Chen, Y.F. Han & J.D. Liang, sp. nov. MycoBank No: 856389 Fig. 8 Etymology. Referring to its location, Bala Valley, where the fungus was first dis- covered. Mycokeys 112: 335-359 (2025), DOI: 10.3897/mycokeys.112.140799 350 Wan-Hao Chen et al.: Four new araneogenous species and a new genus in Hypocreales Type. CHINA * Guizhou Province, Guiyang City, Wudang District, Bala Valley (26°45'7.0344"N, 106°58'57.09"E). On a dead spider (Araneae), 5 April 2024, Wanhao Chen, GZAC WD0410 (holotype), ex-type, WD04101. Description. Colonies on PDA reaching 25-36 mm diam in 14 d at 25 °C, white, consisting of a basal felt and cottony, floccose overgrowth, reverse yel- lowish, with radial patterns. Prostrate hyphae smooth, septate, hyaline, 1.5- 2.0 um diam. Conidial structures consisting of erect conidiophores usually arising from the aerial hyphae, solitary or lecanicillium-like with conidiogenous cells in whorls of two to four. Conidiogenous cells 3.8-5.4 x 1.3-1.9 um, with a cylindrical basal portion, bearing numerous denticles, tapering into a distinct neck. Conidia hyaline, ellipsoidal to fusiform, 1-celled, 2.3-3.0 x 1.7-1.9 um. Octahedral crystals not observed. Host. Spider (Araneae). Additional material examined. CHINA * Guizhou Province, Guiyang City, Wu- dang District, Bala Valley (26°45'7.0344'"N, 106°58'57.09"E). On a dead spider (Araneae), 5 April 2024, Wanhao Chen, WD04102 (living culture). Remarks. Gamszarella vallensis was identified as in Gamszarella, based on the BLASTn results in NCBI. The phylogenetic analysis of the combined dataset 2 (Fig. 2) showed that the new strains clustered into an independent clade with a close relationship with Gamszarella antillana and G. buffelskloofina. Compared with the typical characteristics of the known species (Table 3), Gamszarella val- lensis was distinguished from G. antillana by its shorter conidiogenous cells bearing numerous denticles (3.8-5.4 x 1.3-1.9 um vs. 18-31 x 1 um), ellipsoidal to fusiform conidia and absent of octahedral crystals. Gamszarella vallensis was Figure 8. Gamszarella vallensis A infected spider B, C pda culture plate showing top (B) and reverse (C) sides of the colony D-J conidiogenous cells and conidia were stained with cotton blue K, L conidiogenous cells and conidia. Scale bars: 10 mm (B, C); 10 um (D-L). MycoKeys 112: 335-359 (2025), DOI: 10.3897/mycokeys.112.140799 351 Wan-Hao Chen et al.: Four new araneogenous species and a new genus in Hypocreales distinguished from G. buffelskloofina by its shorter conidiogenous cells (3.8-5.4 x 1.3-1.9 um vs. 7-22 x 1.5-2 um), smaller ellipsoidal to fusiform conidia [2.3- 3.0 x 1.7-1.9 um vs. (3-)4-6(-10) x 2 um] and spider host. Gamszarella vallensis was distinguished from G. sinensis by its shorter conidiogenous cells (3.8—5.4 x 1.3-1.9 um vs. 7.0-12.0 x 1.0-1.5 pm). Thus, the morphological characteristics and molecular phylogenetic results support G. vallensis as a new species. Neogamszarella W.H. Chen, Y.F. Han & J.D. Liang, gen. nov. MycoBank No: 856411 Etymology. Named after its morphological similarity to the genus Gamszarella. Type species. Neogamszarella antillana (R.F. Castafieda & G.R.W. Arnold) W.H. Chen, Y.F. Han & J.D. Liang (Basionym = Verticillium antillanum R.F. Castafieda & G.R.W. Arnold, Feddes Repert. Spec. Nov. Regni Veg. 98 (7-8): 411, 1987). Description. Colonies on PDA, white, with cream-colored reverse, without diffusing pigment into the agar. Conidiogenous cells developing on prostrate hyphae, single or up to 6 in verticils, subulate. Conidia solitary and of two types. Octahedral crystals present. Host. Agaric (Hymenomycetes). Sexual morph. Unknown. Remarks. The genus Gamszarella was established with the type species G. buffelskloofina and two species, G. antillana and G. magnispora (Z.F. Zhang & L. Cai) Crous based on the phylogenetic analysis (Crous et al. 2023). Khonsanit et al. (2024) introduced a new genus Corpulentispora Khons., Thanakitp. & Lu- angsa-ard to accommodate Gamszarella magnispora based on the phylogenet- ic analysis and morphological characteristics. The morphological characteris- tics of Gamszarella buffelskloofina are conidiogenous cells subcylindrical with apical taper, terminating in a cluster of denticles and only one type of subcy- lindrical conidia (Crous et al. 2023). While the morphological characteristics of Gamszarella antillana are conidiogenous cells absent of denticles and produce two types of fusoid conidia, macroconidia and microconidia. G. antillana do not fit with the genus Gamszarella. Thus, a new genus is proposed to accommo- date species Gamszarella antillana. Neogamszarella antillana (R.F. Castaheda & G.R.W. Arnold) W.H. Chen, Y.F. Han & J.D. Liang, comb. nov. MycoBank No: 856412 = Verticillium antillanum R.F. Castaneda & G.R.W. Arnold, Feddes Repert. Spec. Nov. Regni Veg. 98 (7-8): 411, 1987. Basionym. = Lecanicillium antillanum (R.F. Castafieda & G.R.W. Arnold) Zare & W. Gams, Nova Hedwigia 73(1—2): 34, 2001. = Gamszarella antillana (R.F. Castafieda & G.R.W. Arnold) Crous, Persoonia 51: 3912023. Remarks. Verticillium antillanum was transferred to the genus Lecanicillium by Zare and Gams (2001). Crous et al. (2023) introduced a new genus Gamszarella MycoKeys 112: 335-359 (2025), DOI: 10.3897/mycokeys.112.140799 359 Wan-Hao Chen et al.: Four new araneogenous species and a new genus in Hypocreales and combined Lecanicillium antillanum with Gamszarella based on the phyloge- netic analysis. The morphological characteristics of Gamszarella antillana were similar to Corniculantispora aranearum (Petch) Khons. et al., C. dimorpha (J.D. Chen) Khons. et al., C. psalliotae (Treschew) Khons. et al., Flavocillium acero- sum (Zare & W. Gams) H. Yu et al., and both species produce two types of co- nidia (Wang et al. 2020; Khonsanit et al. 2024). However, Gamszarella antillana was distinguished from Corniculantispora aranearum, C. dimorpha, C. psalliotae and Flavocillium acerosum by the phylogenetic analysis (Fig. 2). Besides, the morphological characteristics of Gamszarella antillana do not fit with the typi- cal characteristics of the genus Gamszarella. Thus, Gamszarella antillana was combined into the new genus Neogamszarella as Neogamszarella antillana. Discussion Karst regions in southwestern China are one of the world’s 36 biodiversity hotspots, home to a wide range of endemic species (Delgado-Baquerizo et al. 2020). Wijayawardene et al. (2021) discussed the necessity of systematic stud- ies to reveal novel taxa in Yunnan-—Guizhou Plateau. Many new entomopatho- genic fungi were found in the Kasit regions of Yunnan and Guizhou Provinces (Peng et al. 2023, 2024; Tang et al. 2023a, b; Xiao et al. 2023, 2024; Zhang et al. 2023; Chen et al. 2024; Dai et al. 2024; Fan et al. 2024; Wang et al. 2024b). Besides, there is high spider diversity in the Karst regions, especially in caves (Zhang and Li 2014; Liu et al. 2023). The present study introduces four new species of Chlorocillium and Gamszarella from spiders. Chlorocillium species are often found on spiders, aphids and scale insects (Zare and Gams 2016). The taxonomic delimitation of Chlorocillium was originally based on morphological characteristics and phylogenetic analysis of ITS or LSU sequences (Zare and Gams 2016). Tan and Shivas (2023, 2024) reported two new species based on phylogenetic analysis of ITS, LSU, RPB2, and tef-1a sequences. In this study, we introduce two new species of Chlorocillium viz., C. guizhouense and C. vallense, based on both morpho-molecular data (ITS, LSU and tef-1a) (Figs 1, 5, 6). Moreover, PHI test was carried out to visualize the differences among Chlorocillium spe- cies, and supports the results of morphological characteristics and phyloge- netic analysis (Fig. 3). Therefore, combined analysis of morphological charac- teristics, phylogenetic analysis and other methods may benefit the taxonomy of Chlorocillium. Crous et al. (2023) introduced Gamszarella with the type species G. buffelsk- loofina and transferred Lecanicillium antillanum (Castafheda & G. Arold) Zare & W. Gams and L. magnisporum Z.F. Zhang & L. Cai into Gamszarella based on the phylogenetic analysis and morphological characteristics. Khonsanit et al. (2024) proposed a new genus Corpulentispora Khons. et al. to accommodate Gamszarella magnispora. |n the present study, two new species of Gamszarella viz., G. sinensis and G. vallensis, are introduced based on both morpho-molec- ular data (ITS, LSU, RPB2 and tef-1a) (Figs 2, 7, 8). However, the morphological characteristics of Gamszarella antillana were significantly different from G. buf- felskloofina, G. sinensis and G. vallensis by its two types of conidia, no denti- cles, presence of octahedral crystals and Agaric substrate. Thus, we proposed a new genus, Neogamszarella, to accommodate this species. MycoKeys 112: 335-359 (2025), DOI: 10.3897/mycokeys.112.140799 353 Wan-Hao Chen et al.: Four new araneogenous species and a new genus in Hypocreales Our study confirms the high fungal diversity associated with arthropods in South-Western China. Nevertheless, fungi associated with spiders are poorly known and need thorough, systematic exploration. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Funding This work was funded by National Natural Science Foundation of China (31860002, 81960692), High-level Innovative Talents Training Object in Guizhou Province (Qianke- hepingtairencai [2020]6005), Construction Program of Key Laboratory of Guizhou Prov- ince (Qiankehepingtairencai-ZDSYS[2023]004), Research Center Project of Guizhou Uni- versity of Traditional Chinese Medicine (Guizhongyi ZX hezi [2024]021). Author contributions Data curation: WHC. Formal analysis: JDL. Funding acquisition: WYT, YFH, JHZ, WHC. Methodology: YFH. Resources: WHC. Writing - original draft: HLS, WHC, JDL, DL. Writing - review and editing: WYT, YFH, JHZ. Author ORCIDs Wan-Hao Chen ® https://orcid.org/0000-0001-7240-6841 Dan Li © https://orcid.org/0009-0009-0256-6749 Hui-Lin Shu ® https://orcid.org/0009-0003-1905-595X Jian-Dong Liang ® https://orcid.org/0000-0002-3939-3900 Jie-Hong Zhao © https://orcid.org/0000-0003-2972-382X Wei-Yi Tian © https://orcid.org/0000-0001-7365-2796 Yan-Feng Han ® https://orcid.org/0000-0002-8646-3975 Data availability All of the data that support the findings of this study are available in the main text or Supplementary Information. References Bruen TC, Philippe H, Bryant D (2006) A simple and robust statistical test for detecting the presence of recombination. 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Supplementary material 1 The genera of araneogenous fungi in the order Hypocreales Authors: Wan-Hao Chen, Dan Li, Hui-Lin Shu, Jian-Dong Liang, Jie-Hong Zhao, Wei-Yi Tian, Yan-Feng Han 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.112.140799.suppl1 MycoKeys 112: 335-359 (2025), DOI: 10.3897/mycokeys.112.140799 359