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Research Article
Chlorocillium sinense sp. nov. (Clavicipitaceae) and Calcarisporium guizhouense sp. nov. (Calcarisporiaceae) in Hypocreales from China
expand article infoWan-Hao Chen‡§|, Dan Li|, Jian-Dong Liang|§, Xiu-Xiu Ren|, Jie-Hong Zhao|, Yan-Feng Han‡
‡ Guizhou University, Guiyang, China
§ Key Laboratory of Microbio and Infectious Disease Prevention & Control in Guizhou Province, Guiyang, China
| Guizhou University of Traditional Chinese Medicine, Guiyang, China

Corresponding author: Yan-Feng Han ( swallow1128@126.com )

Academic editor: Nalin Wijayawardene
© 2024 Wan-Hao Chen, Dan Li, Jian-Dong Liang, Xiu-Xiu Ren, Jie-Hong Zhao, Yan-Feng Han.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Chen W-H, Li D, Liang J-D, Ren X-X, Zhao J-H, Han Y-F (2024) Chlorocillium sinense sp. nov. (Clavicipitaceae) and Calcarisporium guizhouense sp. nov. (Calcarisporiaceae) in Hypocreales from China. MycoKeys 109: 91-107. https://doi.org/10.3897/mycokeys.109.128060
Open Access

Abstract

Two new species, Chlorocillium sinense and Calcarisporium guizhouense, isolated from a spider and fruiting body of Cordyceps sp., are introduced. Morphological comparisons and phylogenetic analyses based on multigene datasets (ITS+LSU+RPB2+tef-1alpha) support the establishment of the new species. A combined dataset of ITS, LSU, RPB2, and tef-1alpha showed the taxonomic placement of Chlorocillium in Clavicipitaceae for the first time. Pseudometarhizium is regarded as a synonym of Chlorocillium and two Pseudometarhizium species are transferred into the latter based on the phylogenetic analysis and morphological characteristics.

Key words

Entomopathogenic fungi, morphology, phylogenetic analysis, Sordariomycetes, taxonomic placement

Introduction

During a survey of fungi associated with insects and spiders from Southwest China, a new spider-associated species and a new hyperparasitic species were found. The morphological characteristics and BLAST results revealed that the new collections belong to Chlorocillium and Calcarisporium. The genus Chlorocillium Zare & W. Gams was proposed to accommodate the species in Verticillium section Albo-erecta, and Chlorocillium griseum (Petch) Zare & W. Gams was described as the type species (Zare and Gams 2016). The typical characteristics of Chlorocillium are its slow-growing and greenish-ochraceous colony, conidiophores short with meagre whorls of phialides and fusiform conidia in chains (Zare and Gams 2016). The genus comprises the type species and two other species, C. gueriniae Y.P. Tan et al. and C. montefioreae Y.P. Tan et al. (Tan and Shivas 2023, 2024).

The genus Calcarisporium Preuss was introduced with C. arbuscula Preuss as the type species (Preuss 1851). The typical characteristics of Calcarisporium are its verticillate conidiophores, holoblastic conidiogenous cells, polyblastic, sympodial conidiation and ovoid to ellipsoidal conidia (Hughes 1951, de Hoog 1974). Sun et al. (2017) accepted six species i.e., C. acerosum Matsush., C. arbuscula, C. cordycipiticola Jing Z. Sun et al., C. ovalisporum (Petch) de Hoog, C. phaeopodium Somrith. & E.B.G. Jones and C. xylariicola Jing Z. Sun et al. Later, Zhu et al. (2023) introduced C. yuanyangense H. Yu bis & J.Y. Zhu.

In our phylogenetic analyses of combined ITS, LSU, RPB2 and tef1-α sequences, Chlorocillium species clustered in Clavicipitaceae (Hypocreales, Hypocreomycetidae) with strong statistical support. Thus, we propose that Chlorocillium belongs to the family Clavicipitaceae. The new collections are phylogenetically and morphologically distinct from other known species. Thus, we introduce Chlorocillium sinese sp. nov. and Calcarisporium guizhouense sp. nov. Besides, Pseudometarhizium species groups with Chlorocillium s. str., and thus, we propose to synonymise Pseudometarhizium under Chlorocillium based on the phylogenetic analyses.

Materials and methods

Specimen collection and identification

The specimens were collected from Monkey-Ear Tiankeng (27°5'12.138"N, 107°0'48.42"E), Kaiyang County, Guiyang and Mayao River Valley (26°22'8.3748"N, 107°23'16.96"E), Duyun City, Qiannan Buyei and Miao Autonomous Prefecture, Guizhou Province, on 19 July 2023 and 1 May 2022. 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 arthropod body was rinsed with sterile water, followed by sterilization with 75% ethanol for 3–5 s and rinsing again three times with sterilized water. After drying on sterilized filter paper, a piece of the synnemata, mycelium or sclerotia 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/l tetracycline (Chen et al. 2019). After fungal colonies emerged from the inoculated 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 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 morphology was determined on PDA cultures incubated at 25 °C for 14 days and the growth rate, the presence of octahedral crystals and colony colors (surface and reverse) were observed. To investigate the microscopic characteristics, a little of the mycelia was picked up from the colony and mounted in lactophenol cotton blue or 20% lactic acid solution and the asexual morphological characteristics (e.g., conidiophores, phialides and conidia) 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 transcribed spacer (ITS) region (White et al. 1990), LR0R/LR5 for 28s large subunit ribosomal (LSU) (Vilgalys and Hester 1990), fRPB2-5F/fRPB2-7cR for RNA polymerase II second largest subunit (RPB2) (Liu et al. 1999) and 983F/2218R for translation elongation factor 1 alpha (tef-1α) (Castlebury et al. 2004). The thermal cycle of PCR amplification for these phylogenetic markers was set up following the procedure described by Chen et al. (2021). PCR products were purified and sequenced at Sangon Biotech (Shanghai) Co. All newly generated sequences were deposited in GenBank and accession numbers were obtained (Table 1).

Table 1.
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List of strains and GenBank accession numbers of sequences used in this study.

Species Strain No. GenBank Accession No.
ITS LSU RPB2 tef–1α
Aciculosporium oplismeni MAFF 246966 LC571760 LC571760 LC572054 LC572040
Aciculosporium take MAFF 241224 LC571753 LC571753 LC572048 LC572034
Aciculosporium take TNS-F-60465 LC571755 LC571756 LC572049 LC572035
Akanthomyces aculeatus HUA 772 KC519371 – – KC519366
Aschersonia confluens BCC 7961 JN049841 DQ384947 DQ452465 DQ384976
Aschersonia placenta BCC 7869 JN049842 EF469074 EF469104 EF469056
Atkinsonella hypoxylon B4728 – – KP689514 KP689546
Balansia epichloe A.E.G. 96-15a – – EF468908 EF468743
Balansia henningsiana A.E.G. 96-27a JN049815 AY545727 DQ522413 AY489610
Balansia pilulaeformis A.E.G. 94-2 – AF543788 DQ522414 DQ522319
Bionectria ochroleuca AFTOL-ID 187 – DQ862027 DQ862013 DQ862029
Bionectria vesiculosa HMAS 183151T HM050304 HM050302 – –
Calcarisporium arbuscula CBS 221.73 AY271809 – – –
Calcarisporium arbuscula CBS 900.68 KT945003 KX442598 KX442597 KX442596
Calcarisporium cordycipiticola CGMCC 3.17905 KT944999 KX442599 KX442594 KX442593
Calcarisporium cordycipiticola CGMCC 3.17904 KT945001 KX442604 KX442607 KX442605
Calcarisporium guizhouense DY05041T PP124948 PP133530 – PP146564
Calcarisporium guizhouense DY05042 PP809658 PP809662 – PP823899
Calcarisporium xylariicola HMAS 276836T KX442603 KX442601 KX442606 KX442595
Calonectria ilicicola CBS 190.50 GQ280605 GQ280727 KM232307 AY725726
Cephalosporium curtipes CBS 154.61 AJ292404 AF339548 EF468947 EF468802
Chlorocillium griseum CBS 387.73T KU382150 KU382218 – –
Chlorocillium griseum RCEF6632 MW031768 MW084342 MW091329 MW091327
Chlorocillium gueriniae BRIP 72680aT OR750699 OR731505 OR737788 OR737799
Chlorocillium gueriniae BRIP 72666a OR750701 OR731507 OR737790 OR737801
Chlorocillium gueriniae BRIP 72668a OR750702 OR731508 OR737791 OR737802
Chlorocillium montefioreae BRIP 70299aT PP420202 PP415875 PP438395 PP438400
Chlorocillium sinense KY07181T PP768154 PP768156 PP766578 PP766580
Chlorocillium sinense KY07182 PP768155 PP768157 PP766579 PP766581
Claviceps fusiformis ATCC 26019 JN049817 U17402 – DQ522320
Claviceps purpurea GAM 12885 U57669 AF543789 DQ522417 AF543778
Claviceps purpurea S.A. cp11 – EF469075 EF469105 EF469058
Clonostachys rosea GJS90-227 – AY489716 – AY489611
Collarina aurantiaca FMR 11134 KJ807178 KJ807181 – –
Collarina aurantiaca FMR 11784 KJ807177 KJ807180 – –
Conoideocrella luteorostrata NHJ 11343 JN049859 EF468850 – EF468801
Conoideocrella luteorostrata NHJ 12516 JN049860 EF468849 EF468946 EF468800
Conoideocrella tenuis NHJ 6293 JN049862 EU369044 EU369087 EU369029
Cocoonihabitus sinensis HMAS254523T KY924870 KY924869 – –
Cocoonihabitus sinensis HMAS254524 MF687395 MF687396 – –
Corallocytostroma ornithocopreoides WAC 8705 – – LT216620 LT216546
Cordyceps brongniartii BCC16585 JN049867 JF415967 JF415991 JF416009
Cordyceps militaris OSC93623 JN049825 AY184966 – DQ522332
Dactylonectria alcacerensis CBS 129087 JF735333 KM231629 – JF735819
Dussiella tuberiformis* – – JQ257020 JQ257027
Elaphocordyceps ophioglossoides NBRC 106332 JN943322 JN941409 – –
Elaphocordyceps paradoxa NBRC 106958 JN943324 JN941411 – –
Ephelis japonica CBS 236.64 MH858427 – – –
Ephelis japonica Eph.oryzae AB038564 – – –
Ephelis tripsaci CBS 857.72T NR_153997 NG_059240 – –
Epichloe elymi C. Schardl 760 – AY986924 – AY986951
Epichloe typhina ATCC 56429 JN049832 U17396 DQ522440 AF543777
Flammocladiella aceris CPC 24422 KR611883 KR611901 – –
Fusarium circinatum CBS 405.97 U61677 – JX171623 KM231943
Fusarium sublunatum CBS 189.34T HQ897830 KM231680 – –
Gelasinospora tetrasperma AFTOL-ID 1287 – DQ470980 DQ470932 DQ471103
Haptocillium sinense CBS 567.95 AJ292417 AF339545 – –
Helicocollum surathaniensis BCC 34463 – KT222328 – KT222336
Helicocollum surathaniensis BCC 34464T – KT222329 – KT222337
Heteroepichloe bambusae Ba-01 AB065426 – – –
Heteroepichloe bambusae Bo-01 AB065428 – – –
Heteroepichloe sasae E. sasae-H AB065432 – – –
Heteroepichloe sasae E. sasae-N AB065431 – – –
Hydropisphaera erubescens ATCC 36093 – AF193230 AY545731 DQ518174
Hydropisphaera lutea ATCC 208838 – AF543791 DQ522446 AF543781
Hydropisphaera peziza GJS92-101 – AY489730 – AY489625
Hydropisphaera rufa DAOM JBT1003 JN942883 JN938865 – –
Hypocrea americana AFTO -ID 52 DQ491488 AY544649 – DQ471043
Hypomyces polyporinus ATCC 76479 – AF543793 – AF543784
Keithomyces carneus CBS 239.32 NR_131993 NG_057769 EF468938 EF468789
Lecanicillium attenuatum CBS 402.78 AJ292434 AF339565 EF468935 EF468782
Lecanicillium lecanii CBS 101247 JN049836 KM283794 KM283859 DQ522359
Lecanicillium psalliotae CBS 367.86 – KM283800 – KM283823
Marquandomyces marquandii CBS 182.27 NR_131994 EF468845 EF468942 EF468793
Marquandomyces sp. CBS 127132 MT078882 MT078857 MT078922 –
Metapochonia bulbillosa CBS 145.70 MH859529 AF339542 EF468943 EF468796
Metapochonia gonioides CBS 891.72 AJ292409 AF339550 DQ522458 DQ522354
Metapochonia rubescens CBS 464.88T – AF339566 EF468944 EF468797
Metapochonia sulchlasporia CBS 251.83 NR_154139 MH873311 – KJ398790
Metarhiziopsis microspora CEHS133a EF464589 EF464571 – –
Metarhiziopsis microspora INEHS133a EF464583 EF464572 – –
Metarhizium anisopliae ARSEF 7487 HQ331446 – DQ468370 DQ463996
Metarhizium anisopliae CBS 130.71T MT078884 MT078853 MT078918 MT078845
Metarhizium flavoviride CBS 125.65 MT078885 MT078854 MT078919 MT078846
Metarhizium flavoviride CBS 700.74 – MT078855 MT078920 MT078847
Metarhizium flavoviride CBS 218.56T MH857590 MH869139 KJ398694 KJ398787
Moelleriella phyllogena CUP 067785 – EU392610 – EU392674
Moelleriella phyllogena CUP 067793 – EU392608 – EU392672
Moelleriella umbospora CUP 067817T – EU392628 – EU392688
Morakotia fusca BCC 64125 – KY794862 – KY794857
Morakotia fusca BCC 79272T – KY794861 – KY794856
Mycophilomyces periconiae CPC 27558 NR_154209 NG_059746 – –
Myriogenospora atramentosa A.E.G 96-32 – AY489733 DQ522455 AY489628
Myrotheciomyces corymbiae CPC 33206 NR_160351 NG_064542 – –
Myrothecium inundatum IMI158855 – AY489731 – AY489626
Myrothecium roridum ATCC 16297 – AY489708 – AY489603
Myrothecium verrucaria ATCC 9095 – AY489713 – AY489608
Nectria cinnabarina CBS 125165 HM484548 HM484562 KM232402 HM484527
Nectria nigrescens CBS 125148 HM484707 HM484720 KM232403 HM484672
Nectriopsis violacea CBS 424.64 – AY489719 – –
Neoaraneomyces araneicola DY101711T MW730520 MW730609 MW753026 MW753033
Neoaraneomyces araneicola DY101712 MW730522 MW730610 MW753027 MW753034
Neobarya parasitica Marson s/n KP899626 KP899626 – –
Neonectria candida CBS 151.29 JF735313 AY677333 – JF735791
Neonectria faginata CBS 217.67 HQ840385 HQ840382 DQ789797 JF268746
Neonectria neomacrospora CBS 118984 HQ840388 HQ840379 DQ789810 JF268754
Neonectria ramulariae CBS 182.36 HM054157 HM042435 DQ789793 HM054092
Neurospora crassa ICMP 6360 AY681193 AY681158 – –
Niesslia exilis CBS 560.74 – AY489720 – AY489614
Nigelia aurantiaca BCC13019 – GU979948 GU979971 GU979957
Nigelia martiale EFCC 6863 – JF415974 – JF416016
Ophiocordyceps heteropoda EFCC 10125 JN049852 EF468812 EF468914 EF468752
Ophiocordyceps sinensis EFCC 7287 JN049854 EF468827 EF468924 EF468767
Ophiocordyceps stylophor OSC 111000 JN049828 DQ518766 DQ522433 DQ522337
Orbiocrella petchii NHJ 6209 JN049861 EU369039 EU369081 EU369023
Orbiocrella petchii NHJ 6240 – EU369038 EU369082 EU369022
Papiliomyces liangshanensis EFCC 1452 – EF468815 – EF468756
Papiliomyces liangshanensis EFCC 1523 – EF468814 EF468918 EF468755
Papiliomyces shibinensis GZUH SB13050311T NR154178 – – KR153589
Parametarhizium changbaiense CGMCC 19143T MN589741 MN589994 MT921829 MN908589
Parametarhizium hingganense CGMCC 19144 MN055703 MN061635 MT939494 MN065770
Paraneoaraneomyces sinensis ZY 22.006 OQ709254 OQ709260 OQ719621 OQ719626
Paraneoaraneomyces sinensis ZY 22.007 OQ709255 OQ709261 OQ719622 OQ719627
Paraneoaraneomyces sinensis ZY 22.008T OQ709256 OQ709262 OQ719623 OQ719629
Parepichloe cinerea Ne-01 AB065425 – – –
Peethambara spirostriata CBS110115 – AY489724 EF692516 AY489619
Periglandula ipomoeae IasaF13 – – KP689517 KP689568
Pochonia boninensis JCM 18597 AB709858 AB709831 AB758693 AB758463
Pochonia chlamydosporia CBS 101244 JN049821 DQ518758 DQ522424 DQ522327
Pseudometarhizium araneogenum DY101741 MW730532 MW730618 MW753030 MW753037
Pseudometarhizium araneogenum DY101742 MW730534 MW730619 MW753031 MW753038
Pseudometarhizium lepidopterorum SD05361T MW730543 MW730624 – MW753041
Pseudometarhizium lepidopterorum SD05362 MW730611 MW730629 – MW753042
Purpureomyces maesotensis BCC 88441 MN781916 MN781877 MN781824 MN781734
Purpureomyces maesotensis BCC 85349 MN781928 MN781872 – MN781729
Purpureomyces maesotensis BCC 89300T MN781917 MN781876 – MN781733
Regiocrella camerunensis ARSEF 7682 – DQ118735 – DQ118743
Rosasphaeria moravica LMM JF440985 – JF440986 JF440987
Rotiferophthora angustispora CBS 101437 AJ292412 AF339535 DQ522460 AF543776
Roumegueriella rufula CBS 346.85 – DQ518776 DQ522461 DQ522355
Roumegueriella rufula GJS 91-164 – EF469082 EF469116 EF469070
Samuelsia chalalensis CUP 067856T – EU392637 – EU392691
Samuelsia mundiveteris BCC 40021 – GU552152 – GU552145
Samuelsia rufobrunnea CUP 067858T – AY986918 – AY986944
Sarocladium bacillisporum CBS 425.67 NR_145039 MH870718 – –
Sarocladium dejongiae CBS 144929T NR_161153 NG_067854 – –
Sarocladium implicatum CBS 959.72T HG965023 MH878470 – –
Sarocladium subulatum CBS 217.35 MH855652 NG_070566 – –
Sarocladium terricola CBS 243.59 MH857853 MH869389 – –
Shimizuomyces paradoxus EFCC 6279 JN049847 EF469084 EF469117 EF469071
Shimizuomyces paradoxus EFCC 6564 – EF469083 EF469118 EF469072
Simplicillium lamellicola CBS 116.25T AJ292393 MH866307 DQ522462 DQ522356
Simplicillium lanosoniveum CBS 101267 AJ292395 – DQ522463 DQ522357
Simplicillium lanosoniveum CBS 704.86 AJ292396 AF339553 DQ522464 DQ522358
Sordaria fimicola AFTOL-ID 216 DQ518178 – – DQ518175
Stachybotrys eucylindrospora ATCC 18851 JN942887 JN938869 – –
Sphaerostilbella aureonitens GJS74-87 FJ442633 HM466683 FJ442763 –
Sphaerostilbella berkeleyana GJS82-274 – U00756 – AF543783
Sphaerostilbella chlorohalonata DAOM 235557 JN942888 JN938870 – –
Stachybotrys microspora CBS 186.79 – – DQ676580 DQ676604
Stephanonectria keithii GJS92-133 – AY489727 – AY489622
Sungia yongmunensis EFCC 2131T JN049856 EF468833 – EF468770
Sungia yongmunensis EFCC 2135 – EF468834 – EF468769
Tilachlidium brachiatum CBS 506.67 KM231839 HQ232177 KM232415 KM231976
Tilachlidium brachiatum CBS 363.97 KM231838 KM231719 KM232414 KM231975
Tolypocladium inflatum SCALT1007-002 KC963032 – – –
Trichoderma aggressivum CBS100525 – JN939837 JQ014130 –
Trichoderma viride GJS89-127 – AY489726 – AY489621
Trichothecium roseum DUCC 502 JN937590 JX458860 – –
Tyrannicordyceps fratricida TNS-F 19011 JQ349068 JQ257023 JQ257021 JQ257028
Ustilaginoidea dichromonae MRL IB9228 – – JQ257018 JQ257025
Ustilaginoidea virens ATCC 16180 – – JQ257019 JQ257026
Ustilaginoidea virens MAFF 240421 – JQ257011 JQ257017 JQ257024
Yosiokobayasia kusanagiensis TNS-F18494 – JF415972 – JF416014
Pleurocordyceps aurantiaca MFLUCC 17-2113 MG136916 MG136910 MG136870 MG136875
Pleurocordyceps marginaliradians MFLU 17-1582T MG136920 MG136914 MG271931 MG136878

Note: * J.F. White, Scale on Arundinaria tecta, North Carolina, 2000. New strains or species are in bold type.“T” denotes ex-type. Abbreviations for collections: 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.

Sequence alignments and phylogenetic analyses

DNASTAR™ Lasergene (v 6.0) was used to edit DNA sequences in this study. The ITS, LSU, RPB2 and tef-1α sequences were downloaded from GenBank, based on Sun et al. (2017), Chen et al. (2022a), Zhang et al. (2023), Zhu et al. (2023), and Tan and Shivas (2023, 2024) and others selected based on BLASTn searches in GenBank. ITS sequences and other loci were aligned and edited by MAFFT v.7.037b (Katoh and Standley 2013) and MEGA6 (Tamura et al. 2013). Combined sequences of ITS, LSU, RPB2 and tef-1α were obtained using 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 combined dataset of ITS, LSU, RPB2 and tef-1α sequence data (analysis 1 and analysis 2) was analyzed using Bayesian inference (BI) and maximum likelihood (ML) methods. For BI, a Markov 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. After the analysis was finished, each run was examined 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 constructed with IQ-TREE (v 2.0) (Trifinopoulos et al. 2016), using an automatic selection of the model according to BIC.

Results

Phylogenetic analyses

The phylogenetic tree (Fig. 1) of analysis 1 was generated to determine the family placement of the new strains and Chlorocillium species. Gelasinospora tetrasperma Dowding (AFTOL-ID 1287), Neurospora crassa Shear & B.O. Dodge (ICMP 6360) and Sordaria fimicola (Roberge ex Desm.) Ces. & De Not. (AFTOL-ID 216) were used as the outgroup taxa in analysis 1. The concatenated sequences of analysis 1 included 79 taxa and consisted of 3,126 (ITS, 633; LSU, 842; RPB2, 846; and tef-1α, 805) characters with gaps. The phylogenetic tree (Fig. 2) of analysis 2 was generated to determine the establishment of Chlorocillium species in Clavicipitaceae (Suppl. material 1). Pleurocordyceps aurantiaca (Y.P. Xiao, T.C. Wen & K.D. Hyde) Y.H. Wang, et al. (MFLUCC 17-2113) and Pleurocordyceps marginaliradians (Y.P. Xiao, T.C. Wen & K.D. Hyde) Y.H. Wang, et al. (MFLU 17-1582) were used as the outgroups in analysis 2. The concatenated sequences of analysis 2 included 69 taxa and consisted of 3,120 (ITS, 671; LSU, 740; RPB2, 825; and tef-1α, 884) characters with gaps.

Figure 1. 

Phylogenetic analysis of the new strains and Chlorocillium species in the order Hypocreales based on multigene dataset (ITS, LSU, RPB2 and tef-1α). Statistical support values (≥ 50%/0.5) are shown at the nodes for ML bootstrap support/BI posterior probabilities. The new taxa are in bold.

Figure 2. 

Phylogenetic analysis of Chlorocillium species in Clavicipitaceae, based on multigene dataset (ITS, LSU, RPB2 and tef-1α). Statistical support values (≥ 50%/0.5) are shown at the nodes for ML bootstrap support/BI posterior probabilities. The new taxa are in bold.

Analysis 1: The selected model for ML analysis was TIM2+F+I+G4. The final value of the highest scoring tree was –46,827.254, which was obtained from an ML analysis of the dataset (ITS+LSU+RPB2+ tef-1α). The parameters of the rate heterogeneity model used to analyze the dataset were estimated using the following frequencies: A = 0.236, C = 0.274, G = 0.278, T = 0.211; substitution rates AC = 1.44290, AG = 2.23422, AT = 1.44290, CG = 1.00000, CT = 5.74279 and GT = 1.00000, as well as the gamma distribution shape parameter α = 0.630. The selected models for BI analysis were GTR+F+I+G4 (ITS, LSU and RPB2), and GTR+F+G4 (tef-1α). The phylogenetic trees (Fig. 1) constructed using ML and BI analyses were largely congruent and strongly supported in most branches. The new strains DY05041 and DY05042 clustered into a group of the family Calcarisporiaceae and have a close relationship with Calcarisporium arbuscula (CBS 221.73 and CBS 900.68) and C. yuanyangense (YFCC 22099256). Strains KY07181, KY07182 and Chlorocillium species clustered as an independent clade and belong to the family Clavicipitaceae. Moreover, Pseudometarhizium araneogenum W.H. Chen, et al. (SD05361 and SD05362) and P. lepidopterorum W.H. Chen, et al. were grouped in Chlorocillium s. str. clade.

Analysis 2: The selected model for ML analysis was TN+F+I+G4. The final value of the highest scoring tree was –41,817.340, which was obtained from the ML analysis of the dataset (ITS+LSU+RPB2+ tef-1α). The parameters of the GTR model used to analyze the dataset were estimated based on the following frequencies: A = 0.233, C = 0.280, G = 0.277, T = 0.210; substitution rates AC = 1.00000, AG = 2.55486, AT = 1.00000, CG = 1.00000, CT = 5.56065 and GT = 1.00000, as well as the gamma distribution shape parameter α = 0.442. The selected models for BI analysis were GTR+F+I+G4 (ITS+LSU+ tef-1α) and SYM+G4 (RPB2). The phylogenetic trees (Fig. 2) constructed using ML and BI analyses were largely congruent and strongly supported in most branches. Most genera clustered into independent clades. Strains KY07181 and KY07182 clustered into an independent clade with close relationship with Chlorocillium griseum. However, Pseudometarhizium species clustered within the genus Chlorocillium.

Taxonomy

Calcarisporium guizhouense W.H. Chen, Y.F. Han & J.D. Liang, sp. nov.

MycoBank No: 854035
Fig. 3

Etymology

Referring to its type location in Guizhou Province.

Type

China • Guizhou Province, Qiannan Buyei and Miao Autonomous Prefecture, Duyun City, Mayao River Valley (26°22'8.3748"N, 107°23'16.96"E), on Cordyceps sp., 1 May 2022, Wanhao Chen, GZAC DY0504 (holotype), ex-type DY05041.

Description

Colonies on PDA attaining a diameter of 28–29 mm after 14 days at 25 °C, white, consisting of a basal felt, floccose hyphal overgrowth, yellowish-white; reverse light brown to brown. Hyphae septate, hyaline, smooth-walled, 2.0–2.2 μm wide. Conidiophores erect, hyaline, verticillately branched, with 1–3 conidiogenous cells. Conidiogenous cells 15.6–23.2 × 1.5–1.7 μm, hyaline, cylindrical at base, gradually tapering near the apex, holoblastic to polyblastic, sympodial, apically with a cluster of conidium-bearing denticles. Conidia 5.2–8.6 × 1.8–2.2 μm, hyaline, smooth-walled, thin-walled, cylindrical, unicellular, acuminate.

Figure 3. 

Calcarisporium guizhouense A the substrate Cordyceps sp. (fungicolous) B, C PDA culture plate showing top (B) and reverse (C) sides of the colony D–K Conidiogenous cells and conidia. Scale bars: 10 mm (B, C); 10 μm (D–K).

Substrate

Cordyceps sp.

Additional strain examined

China • Guizhou Province, Qiannan Buyei and Miao Autonomous Prefecture, Duyun City, Mayao River Valley (26°22'8.3748"N, 107°23'16.96"E). On Cordyceps sp., 1 May 2022, Wanhao Chen, DY05042 (living culture).

Notes

Calcarisporium guizhouense was easily identified as Calcarisporium, based on the BLASTn result in NCBI and its verticillate conidiophores and sympodial conidiation. Phylogenetic analyses show that Calcarisporium guizhouense has close relationships to C. arbuscula and C. yuanyangense (Fig. 1). However, C. guizhouense was easily distinguished from C. arbuscula (Conidia: ovoid to ellipsoid, 4–11× 1.8–3.3 μm; substrate: decaying agaric) and C. yuanyangense (Conidia: ovoid to ellipsoid, 6.33–9.68 × 1.87–2.63 μm; substrate: Ophiocordyceps nutans) by its smaller cylindrical conidia and the substrate. Thus, the morphological characteristics and molecular phylogenetic results support C. guizhouense as a new species.

Chlorocillium Zare & W. Gams, Mycol. Progr. 15: 1005, 2016

= Pseudometarhizium W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang, MycoKeys 91: 59, 2022 MycoBank No: 842641.

Type species

Chlorocillium griseum (Petch) Zare & W. Gams.

Notes

Chen et al. (2022a) introduced the genus Pseudometarhizium with P. araneogenum (type species) and P. lepidopterorum in the family Clavicipitaceae. However, in the present study, the multi-gene phylogenetic analyses revealed that Pseudometarhizium species clustered with Chlorocillium s. str. (Fig. 2). Besides, the typical characteristics of Pseudometarhizium are entirely consistent with Chlorocillium. Thus, the genus Pseudometarhizium is synonymized under Chlorocillium and the two known species of Pseudometarhizium are transferred into Chlorocillium.

Chlorocillium sinense W.H. Chen, Y.F. Han & J.D. Liang, sp. nov.

MycoBank No: 853937
Fig. 4

Etymology

Referring to the country where the fungus was first discovered.

Type

China • Guizhou Province, Guiyang, Kaiyang County, Monkey-Ear Tiankeng (27°5'12.138"N, 107°0'48.42"E), on a dead spider (Araneae), 19 July 2023, Wanhao Chen, GZAC KY0718 (holotype), ex-type, KY07181.

Description

Colonies on PDA reaching 15–17 mm in diameter in 14 days at 25 °C, green to yellowish green in center with white margin, reverse yellowish to light brown. Hyphae septate, hyaline, smooth-walled, 1.3–2.1 μm wide. Conidiophores hyaline, smooth-walled, emerging from aerial hyphae or chondroid mycelium, with single phialide or whorls of 2–4 phialides or verticillium-like from hyphae directly. Phialides cylindrical, somewhat inflated base, 11.7–20.1 × 1.1–1.3 μm, tapering to a thin neck. Conidia hyaline, smooth-walled, fusiform to ellipsoidal, 1.9–2.9 × 0.8–1.2 μm, forming divergent and basipetal chains. Octahedral crystals and chlamydospores absent.

Figure 4. 

Chlorocillium sinense A infected spider B, C PDA-containing culture plate showing B the front and C reverse sides of the colony D–H phialides, conidia in chains and conidia. Scale bars: 10mm (B, C); 10 μm (D–H).

Host

Spider (Araneae).

Additional strain examined

China • Guizhou Province, Guiyang, Kaiyang County, Monkey-Ear Tiankeng (27°5'12.138"N, 107°0'48.42"E). On a dead spider (Araneae), 19 July 2023, Wanhao Chen, KY07182 (living culture).

Remarks

Chlorocillium sinense was easily identified as Chlorocillium, based on the BLASTn result in NCBI. The phylogenetic analysis of the combined dataset of ITS, LSU, RPB2 and tef-1α sequence data showed that the new collections clustered as an independent clade with close relationship to C. araneogenum, C. griseum and C. lepidopterorum (Fig. 2). Table 2 provides the different morphological characteristics that can be used to differentiate Chlorocillium sinense from other phylogenetically close species.

Table 2.
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Morphological comparison of the new species with other Chlorocillium species.

Species Strain Phialides (μm) Conidia (μm) Host Octahedral crystals
C. araneogenum DY101801 8.3–23.3 × 1.3–2.2 fusiform, 3.4–5.8 × 1.4–1.8 spider absent
C. griseum CBS 387.73 18–40(−55) × 2–2.5 fusiform, 4.5–6 × 1.0–1.5 spider present
C. lepidopterorum SD05361 21.2–33.7 × 1.1–1.4 fusiform, 3.1–4.3 × 1.3–1.5 pupa absent
C. sinense KY07181 11.7–20.1 × 1.1–1.3 fusiform to ellipsoidal, 1.9–2.9 × 0.8–1.2 spider absent

New combinations

Chlorocillium araneogenum (W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang) W.H. Chen, Y.F. Han & J.D. Liang, comb. nov.

MycoBank No: 853934

Pseudometarhizium araneogenum W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang, MycoKeys 91: 60 (2022) (Basionym).

Description

Colonies irregular on PDA, 1.8–2.8 cm diam. after 14 d at 25 °C, white, consisting of a basal felt, floccose hyphal overgrowth, reverse yellowish to pale brown or green. Prostrate hyphae smooth, septate, hyaline, 1.0–1.2 μm diam. Erect conidiophores usually arising from aerial hyphae. Phialides solitary or in groups of two, 8.3–23.3 × 1.3–2.2 μm, with a cylindrical basal portion, tapering into a short distinct neck. Conidia in chains, hyaline, fusiform, one-celled, 3.4–5.8 × 1.4–1.8 μm.

Material examined (type material)

China • Duyun City, Qiannan Buyi and Miao Autonomous Prefecture, Guizhou Province. On a dead spider (Araneae), 1 October 2019, Wanhao Chen, GZAC DY10174, living cultures, DY101741, DY101742.

Chlorocillium lepidopterorum (W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang) W.H. Chen, Y.F. Han & J.D. Liang, comb. nov.

MycoBank No: 853935

Pseudometarhizium lepidopterorum W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang, MycoKeys 91: 60 (2022) (Basionym).

Description

Colonies on PDA, 1.4–2.0 cm diam. after 14 d at 25 °C, white, consisting of a basal felt and cottony, floccose hyphal overgrowth, reverse yellowish to pale green. Prostrate hyphae smooth, septate, hyaline, 1.0–2.0 μm diam. Erect conidiophores usually arising from aerial hyphae. Phialides solitary or in groups of two to three, 21.2–33.7 × 1.1–1.4 μm, with a cylindrical basal portion, tapering into a short distinct neck. Conidia in chains, hyaline, fusiform, one-celled, 3.1–4.3 × 1.3–1.5 μm.

Material examined (type material)

China • Sandu County, Qiannan Buyi and Miao Autonomous Prefecture, Guizhou Province, . On a pupa (Lepidoptera), 1 May 2019, Wanhao Chen, GZAC SD0536, living cultures, SD05361, SD05362.

Discussion

The complex terrain, mild climate, abundant rainfall, wide vegetation coverage, and diverse forest types and components, result in Guizhou Province having abundant resources of fungi associated with insects. However, while fungal species associated with insects are often found in forest and grassland reservations (Chen et al. 2019, 2020, Peng et al. 2023, Xiao et al. 2023, 2024, Liu et al. 2024); they are rarely found in special karst eco-environments, such as Tiankeng and valleys. Chen et al. (2022a, b, 2023) introduced thirteen new species associated with insects or spiders from Tiankeng or valleys.

Calcarisporium species are often reported as fungicolous and produce abundant secondary metabolites (Sun et al. 2017, Zhu et al. 2023). Three species, C. cordycipiticola, C. ovalisporum and C. yuanyangense were introduced from the entomopathogenic taxa, Cordyceps militaris (L.) Fr., Hirsutella citriformis Speare and Ophiocordyceps nutans (Pat.) G.H. Sung et al. In the present study, Calcarisporium guizhouense were introduced with the substrate Cordyceps sp. from Mayao River Valley.

Zare and Gams (2016) introduced the genus Chlorocillium with C. griseum, and the placement of the genus was not confirmed in Hypocreomycetidae. Wang (2022) noted that the genus Chlorocillium belongs to the order Hypocreales and has a close relationship with the family Clavicipitaceae. Tan and Shivas (2023, 2024) reported two new Chlorocillium species in Australia. However, the taxonomic status of the genus was still unclear.

In the order-level phylogenetic tree (Fig. 2), Chlorocillium species clustered into Clavicipitaceae (Hypocreales, Hypocreomycetidae, Sordariomycetes). Thus, the combined dataset of ITS, LSU, RPB2, and tef-1α showed the taxonomic placement of Chlorocillium in Clavicipitaceae for the first time. Furthermore, in the family-level phylogenetic tree (Fig. 1), strains KY07181 and KY07182 formed an independent branch and clustered with C. griseum, C. gueriniae, C. montefioreae, P. araneogenum and P. lepidopterorum in a subclade. Pseudometarhizium species were also consistent with the genus Chlorocillium based on the typical characteristics. Thus, we synonymized Pseudometarhizium under Chlorocillium and transferred its species.

Conclusion

Two new species, Chlorocillium sinense and Calcarisporium guizhouense, were established and described in the present study. The genus Chlorocillium was confirmed in the family Clavicipitaceae. Furthermore, the genus Pseudometarhizium was synonymized with Chlorocillium and its species were transferred.

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 High-level Innovative Talents Training Object in Guizhou Province (Qiankehepingtairencai [2020]6005), Science and Technology Foundation of Guizhou Province (No. qiankehejichu-ZK [2022] general482), National Natural Science Foundation of China (31860002, 81960692), Construction Program of Key Laboratory of Guizhou Province (Qiankehepingtairencai-ZDSYS[2023]004), Research Center Project of Guizhou University of Traditional Chinese Medicine (Guizhongyi ZX hezi [2024]021), Construction Program of Guizhou Engineering Research Center (Qian Fa Gai Gao Ji 2020-896).

Author contributions

Data curation: WHC. Formal analysis: DL, JDL, WHC. Funding acquisition: JHZ, WHC, YFH, XXR. Resources: DL, WHC. Writing – original draft: DL, WHC. Writing – review and editing: XXR, JHZ, JDL, YFH.

Author ORCIDs

Wan-Hao Chen https://orcid.org/0000-0001-7240-6841

Jian-Dong Liang https://orcid.org/0000-0002-3939-3900

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.

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Supplementary material

Supplementary material 1 

The genus contain in the family Clavicipitaceae

Wan-Hao Chen, Dan Li, Jian-Dong Liang, Xiu-Xiu Ren, Jie-Hong Zhao, Yan-Feng Han

Data type: docx

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.
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