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Research Article
Phylogenetic, ecological and morphological characteristics reveal two new spider-associated genera in Clavicipitaceae
expand article infoWan-Hao Chen, Jian-Dong Liang, Xiu-Xiu Ren, Jie-Hong Zhao, Yan-Feng Han§, Zong-Qi Liang§
‡ Guizhou University of Traditional Chinese Medicine, Guiyang, China
§ Guizhou University, Guiyang, China
Open Access

Abstract

Clavicipitaceous fungi are pathogenic to scale insects, white flies and other insect orders. However, a few species are spider-associated. Two new genera from China, Neoaraneomyces and Pseudometarhizium, are described based on phylogenetic, ecological and morphological characteristics. Two spider-associated species, Neoaraneomyces araneicola, Pseudometarhizium araneogenum, and an insect-associated species Pseudometarhizium lepidopterorum are included. The morphological characteristics of paecilomyces-like conidiogenous structures, present in many insect/spiders associated species make species-level identifications difficult. A phylogenetic analysis of the combined dataset (ITS, LSU, RPB2 and TEF), placed the two new genera in Clavicipitaceae. The new spider-associated species may be the result of convergent evolution to adapt to the ecological environment and may have undergone host jumping or altered their nutritional preferences.

Keywords

Clavicipitaceae, convergent evolution, morphology, nutritional preference, phylogeny

Introduction

Araneogenous or araneopathogenic fungi are spider-pathogenic fungi (Evans and Samson 1987) and found in diverse habitats, such as different kinds of monocot, dicot or coniferous plants including trees, grasses, bamboo, mosses, ferns, and lichens (Shrestha et al. 2019). The known araneogenous fungal genera include Cordyceps Fr., and the related anamorphic genera Akanthomyces Lebert, Beauveria Vuill., Clathroconium Samson & H.C. Evans, Clonostachys Corda, Gibellula Cavara, Hevansia Luangsa-ard, Hywel-Jones & Spatafora, Hirsutella Pat., Hymenostilbe Petch, Nomuraea Maubl. and Purpureocillium Luangsaard, Hywel-Jones, Houbraken & Samson (Chen et al. 2018). Shrestha et al. (2019) noted that araneogenous fungi are restricted to Cordycipitaceae and Ophiocordycipitaceae, with one exception in Bionectriaceae; there is no report to date of araneogenous fungi in the family Clavicipitaceae within Hypocreales.

Members of Clavicipitaceae are distributed worldwide and found in almost all terrestrial ecosystems. Currently, Clavicipitaceae contains 49 genera and over 500 species (Hyde et al. 2020; Mongkolsamrit et al. 2020; Gao et al. 2021). Among these genera, Claviceps Tul. and Balansia Speg. are pathogenic only to plants (Diehl 1950). Pochonia Bat. & O.M. Fonseca and Rotiferophthora G.L. Barron are pathogenic to a wide variety of invertebrates. Seven sexually reproductive genera, Aschersonia Mont. (Hypocrella), Conoideocrella D. Johnson, G.H. Sung, Hywel-Jones & Spatafora, Orbiocrella D. Johnson, G.H. Sung, Hywel-Jones & Spatafora, Regiocrella P. Chaverri & K.T. Hodge, Samuelsia P. Chaverri & K.T. Hodge and Moelleriella Bres. are pathogenic to scale insects and white flies (Hemiptera), while Metarhizium (Metarcordyceps) has a broad host association (Luangsa-ard et al. 2017).

During a survey of entomopathogenic fungi and their allies in southwestern China, infected insect and spider specimens were obtained, and some fungal strains were isolated and purified. The goal of this research is to identify those new strains by multigene phylogeny, morphological and ecological characteristics.

Materials and methods

Specimen collection and identification

Four infected insect and spider specimens (DY10171, DY10174, DY10180 and SD0536) were collected from Duyun City (26°21'24.71"N, 107°22'48.22"E) and Sandu County (25°57'22.21"N, 107°57'54.69"E), Guizhou Province, on 1 October and 1 May, 2019. Isolation of strains was conducted as described by Chen et al. (2019). Fungal colonies emerging from specimens were isolated and cultured at 25 °C for 14 days under 12 h light/12 h dark conditions following protocols described by Zou et al. (2010). The specimens and the isolated living strains were deposited in the Institute of Fungus Resources, Guizhou University (formally Herbarium of Guizhou Agricultural College; code, GZAC), Guiyang City, Guizhou, China.

Macroscopic and microscopic morphological characteristics of the fungi were examined, especially for the arrangement, shape and measurement of phialides and conidia, and also the growth rates were determined from cultures grown on potato dextrose agar (PDA) cultures incubated at 25 °C for 14 days. Hyphae and conidiogenous structures were mounted in lactophenol cotton blue or 20% lactic acid solution and observed with an optical microscope (OM, DM4 B, Leica, Germany).

DNA extraction, polymerase chain reaction amplification and nucleotide sequencing

DNA extraction was carried out by Fungal genomic DNA Extraction Kit (DP2033, BioTeke Corporation) in accordance with Liang et al. (2011). The extracted DNA was stored at −20 °C. The amplification of internal transcribed spacer (ITS) region, large subunit ribosomal RNA (LSU) gene, RNA polymerase II largest subunit 2 (RPB2) and translation elongation factor 1 alpha (TEF) by PCR was as described by White et al. (1990), Rakotonirainy et al. (1994), Castlebury et al. (2004) and van den Brink et al. (2012), respectively. Primer sequence information is shown in Suppl. material 1. PCR products were purified and sequenced at Sangon Biotech (Shanghai) Co. The resulting sequences were submitted to GenBank (Table 1).

Table 1.

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

Species Strain No. GenBank Accession No.
ITS LSU RPB2 TEF
Aciculosporium oplismeni MAFF 246966 LC571760 LC571760 LC572054 LC572040
A. take MAFF 241224 LC571753 LC571753 LC572048 LC572034
A. take TNS-F-60465 LC571755 LC571756 LC572049 LC572035
Akanthomyces aculeatus HUA 772 KC519371 - - KC519366
Aschersonia badia BCC 8105 - DQ518752 DQ522411 DQ522317
A. placenta BCC 7869 - EF469074 EF469104 EF469056
Atkinsonella hypoxylon B4728 - - KP689514 KP689546
Balansia epichloe A.E.G. 96-15a - - EF468908 EF468743
B. henningsiana GAM 16112 - AY545727 DQ522413 AY489610
B. pilulaeformis A.E.G. 94-2 - AF543788 DQ522414 DQ522319
Bionectria ochroleuca AFTOL-ID 187 - DQ862027 DQ862013 DQ862029
B. vesiculosa HMAS 183151 HM050304 HM050302 - -
Calcarisporium arbuscula CBS 221.73 AY271809 - - -
C. arbuscula CBS 900.68 KT945003 KX442598 KX442597 KX442596
C. cordycipiticola CGMCC 3.17905 KT944999 KX442599 KX442594 KX442593
C. cordycipiticola CGMCC 3.17904 KT945001 KX442604 KX442607 KX442605
C. xylariicola HMAS 276836 KX442603 KX442601 KX442606 KX442595
Calonectria ilicicola CBS 190.50 GQ280605 GQ280727 KM232307 AY725726
Cephalosporium curtipes CBS 154.61 AJ292404 AF339548 EF468947 EF468802
Claviceps fusiformis ATCC 26019 JN049817 U17402 - DQ522320
C. purpurea GAM 12885 - AF543789 DQ522417 AF543778
C. purpurea S.A. cp11 - EF469075 EF469105 EF469058
Clonostachys rosea GJS90-227 - AY489716 - AY489611
Cocoonihabitus sinensis HMAS254523 KY924870 KY924869 - -
C. sinensis HMAS254524 MF687395 MF687396 - -
Collarina aurantiaca FMR 11134 KJ807178 KJ807181 - -
C. aurantiaca FMR 11784 KJ807177 KJ807180 - -
Conoideocrella luteorostrata NHJ 11343 - EF468850 - EF468801
C. luteorostrata NHJ 12516 - EF468849 - EF468800
C. tenuis NHJ 6293 - EU369044 EU369087 EU369029
Corallocytostroma ornithocopreoides WAC 8705 - - LT216620 LT216546
Cordyceps brongniartii BCC16585 JN049867 JF415967 JF415991 JF416009
C. militaris OSC93623 JN049825 AY184966 - DQ522332
Dactylonectria alcacerensis CBS 129087 JF735333 KM231629 - JF735819
Dussiella tuberiformis* - - JQ257020 JQ257027
Elaphocordyceps ophioglossoides NBRC 106332 JN943322 JN941409 - -
E. paradoxa NBRC 106958 JN943324 JN941411 - -
Ephelis japonica CBS 236.64 MH858427 - - -
E. japonica Eph.oryzae AB038564 - - -
E. tripsaci CBS 857.72 NR_153997 NG_059240 - -
Epichloe elymi C. Schardl 760 - AY986924 - AY986951
E. typhina ATCC 56429 JN049832 U17396 DQ522440 AF543777
Flammocladiella aceris CPC 24422 KR611883 KR611901 - -
Fusarium circinatum CBS 405.97 U61677 - JX171623 KM231943
F. sublunatum CBS 189.34 HQ897830 KM231680 - -
Gelasinospora tetrasperma AFTOL-ID 1287 - DQ470980 DQ470932 DQ471103
Haptocillium sinense CBS 567.95 AJ292417 AF339545 - -
Helicocollum krabiensis BCC 71374 - KT222327 - KT222342
H. surathaniensis BCC 34463 - KT222328 - KT222336
H. surathaniensis BCC 34464 - KT222329 - KT222337
Heteroepichloe bambusae Ba-01 AB065426 - - -
H. bambusae Bo-01 AB065428 - - -
H. sasae E. sasae-H AB065432 - - -
H. sasae E. sasae-N AB065431 - - -
Hydropisphaera erubescens ATCC 36093 - AF193230 AY545731 DQ518174
H. lutea ATCC 208838 - AF543791 DQ522446 AF543781
H. peziza GJS92-101 - AY489730 - AY489625
H. rufa DAOM JBT1003 JN942883 JN938865 - -
Hypocrea americana AFTO -ID 52 DQ491488 AY544649 - DQ471043
Hypocrella discoidea BCC 8237 JN049840 DQ384937 DQ452461 DQ384977
Hypomyces polyporinus ATCC 76479 - AF543793 - AF543784
H. aurantius GJS74-69 FJ442642 HM466684 FJ442744 FJ467643
Keithomyces sp. CBS 126563 - MT078856 - MT078921
K. carneus CBS 239.32 NR_131993 NG_057769 EF468938 EF468789
Lecanicillium attenuatum CBS 402.78 AJ292434 AF339565 EF468935 EF468782
L. lecanii CBS 101247 JN049836 KM283794 KM283859 DQ522359
L. psalliotae CBS 367.86 - KM283800 - KM283823
Marquandomyces marquandii CBS 182.27 NR_131994 EF468845 EF468942 EF468793
Marquandomyces sp. CBS 127132 - MT078857 MT078922 -
Metapochonia bulbillosa CBS 145.70 - AF339542 EF468943 EF468796
M. gonioides CBS 891.72 AJ292409 AF339550 DQ522458 DQ522354
M. rubescens CBS 464.88 - AF339566 EF468944 EF468797
M. sulchlasporia CBS 251.83 NR_154139 MH873311 - KJ398790
Metarhiziopsis microspora CEHS133a EF464589 EF464571 - -
M. microspora INEHS133a EF464583 EF464572 - -
Metarhizium anisopliae ARSEF 7487 - - DQ468370 DQ463996
M. anisopliae CBS 130.71 MT078884 MT078853 MT078918 MT078845
M. flavoviride CBS 125.65 MT078885 MT078854 MT078919 MT078846
M. flavoviride CBS 700.74 - MT078855 MT078920 MT078847
M. flavoviride CBS 218.56 - - - KJ398787
Moelleriella phyllogena CUP 067785 - EU392610 - EU392674
M. phyllogena CUP 067793 - EU392608 - EU392672
M. schizostachyi BCC 14123 - DQ518771 DQ522447 DQ522346
M. umbospora CUP 067817 - EU392628 - EU392688
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
M. roridum ATCC 16297 - AY489708 - AY489603
M. verrucaria ATCC 9095 - AY489713 - AY489608
Nectria cinnabarina CBS 125165 HM484548 HM484562 KM232402 HM484527
N. nigrescens CBS 125148 HM484707 HM484720 KM232403 HM484672
Nectriopsis violacea CBS 424.64 - AY489719 - -
Neoaraneomyces araneicola DY101711 MW730520 MW730609 MW753026 MW753033
N. araneicola DY101712 MW730522 MW730610 MW753027 MW753034
Neobarya parasitica Marson s/n KP899626 KP899626 - -
Neonectria candida CBS 151.29 JF735313 AY677333 - JF735791
N. faginata CBS 217.67 HQ840385 HQ840382 DQ789797 JF268746
N. neomacrospora CBS 118984 HQ840388 HQ840379 DQ789810 JF268754
N. 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
N. martiale EFCC 6863 - JF415974 - JF416016
Ophiocordyceps heteropoda EFCC 10125 JN049852 EF468812 EF468914 EF468752
O. sinensis EFCC 7287 JN049854 EF468827 EF468924 EF468767
O. stylophor OSC 111000 JN049828 DQ518766 DQ522433 DQ522337
Orbiocrella petchii NHJ 6209 - EU369039 EU369081 EU369023
O. petchii NHJ 6240 - EU369038 EU369082 EU369022
Papiliomyces liangshanensis EFCC 1452 - EF468815 - EF468756
P. liangshanensis EFCC 1523 - EF468814 EF468918 EF468755
P. shibinensis GZUH SB13050311 NR154178 - - KR153589
Parametarhizium changbaiense CGMCC 19143 MN589741 MN589994 MT921829 MN908589
P. hingganense CGMCC 19144 MN055703 MN061635 MT939494 MN065770
Parepichloe cinerea Ne-01 AB065425 - - -
Peethambara spirostriata CBS110115 - AY489724 EF692516 AY489619
Periglandula ipomoeae IasaF13 - - KP689517 KP689568
Pochonia boninensis JCM 18597 AB709858 AB709831 AB758693 AB758463
P. globispora CBS 203.86 DQ516079 - - -
Pseudometarhizium araneogenum DY101741 MW730532 MW730618 MW753030 MW753037
P. araneogenum DY101742 MW730534 MW730619 MW753031 MW753038
P. araneogenum DY101801 MW730536 MW730623 MW753032 MW753039
P. araneogenum DY101802 MW730545 MW730625 - MW753040
P. lepidopterorum SD05361 MW730543 MW730624 - MW753041
P. lepidopterorum SD05362 MW730611 MW730629 - MW753042
Purpureocillium lavendulum FMR 10376 - FR775489 - FR775516
P. lilacinus CBS 284.36 - - EF468941 EF468792
Purpureomyces maesotensis BCC 88441 MN781916 MN781877 MN781824 MN781734
P. maesotensis BCC 85349 MN781928 MN781872 - MN781729
P. maesotensis BCC 89300 MN781917 MN781876 - MN781733
Regiocrella camerunensis ARSEF 7682 - DQ118735 - DQ118743
Romanoa terricola WCM_17 KP794435 - - -
R. terricola WCM_18 KP794436 - - -
Rosasphaeria moravica LMM JF440985 - JF440986 JF440987
Rotiferophthora angustispora CBS 101437 - AF339535 DQ522460 AF543776
Roumegueriella rufula CBS 346.85 - DQ518776 DQ522461 DQ522355
R. rufula GJS 91-164 - EF469082 EF469116 EF469070
Samuelsia chalalensis CUP 067856 - EU392637 - EU392691
S. mundiveteris BCC 40021 - GU552152 - GU552145
S. rufobrunnea CUP 067858 - AY986918 - AY986944
Sarocladium bacillisporum CBS 425.67 NR_145039 MH870718 - -
S. dejongiae CBS 144929 NR_161153 NG_067854 - -
S. implicatum CBS 959.72 HG965023 MH878470 - -
S. subulatum CBS 217.35 MH855652 NG_070566 - -
S. terricola CBS 243.59 MH857853 MH869389 - -
Shimizuomyces paradoxus EFCC 6279 JN049847 EF469084 EF469117 EF469071
S. paradoxus EFCC 6564 - EF469083 EF469118 EF469072
Simplicillium lamellicola CBS 116.25 AJ292393 MH866307 DQ522462 DQ522356
S. lanosoniveum CBS 101267 AJ292395 - DQ522463 DQ522357
S. 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 -
S. berkeleyana GJS82-274 - U00756 - AF543783
S. chlorohalonata DAOM 235557 JN942888 JN938870 - -
Stachybotrys microspora CBS 186.79 - - DQ676580 DQ676604
Stephanonectria keithii GJS92-133 - AY489727 - AY489622
Sungia yongmunensis EFCC 2131 JN049856 EF468833 - EF468770
S. yongmunensis EFCC 2135 - EF468834 - EF468769
Tilachlidium brachiatum CBS 506.67 KM231839 HQ232177 KM232415 KM231976
T. brachiatum CBS 363.97 KM231838 KM231719 KM232414 KM231975
Tolypocladium inflatum SCALT1007-002 KC963032 - - -
Trichoderma aggressivum CBS100525 - JN939837 JQ014130 -
T. arundinaceum ATCC 90237 EU330927 - EU338326 EU338291
T. viride GJS89-127 - AY489726 - AY489621
Trichosphaerella ceratophora CBS 130.82 KM231847 KM231727 KM232423 KM231983
Trichothecium indicum CBS 123.78 - NG_057651 - -
T. roseum DUCC 502 JN937590 JX458860 - -
Tyrannicordyceps fratricida TNS-F 19011 JQ349068 JQ257023 JQ257021 JQ257028
Ustilaginoidea dichromonae MRL IB9228 - - JQ257018 JQ257025
U. virens ATCC 16180 - - JQ257019 JQ257026
U. virens MAFF 240421 - JQ257011 JQ257017 JQ257026
Valetoniellopsis laxa GJS96-174 - AY015635 AY015638 -
Yosiokobayasia kusanagiensis TNS-F18494 - JF415972 - JF416014

Sequence alignment and phylogenetic analyses

Lasergene software (version 6.0, DNASTAR) was applied for the editing of DNA sequences in this study. The ITS, LSU, RPB2 and TEF sequences were downloaded from GenBank, based on Mongkolsamrit et al. (2018, 2020), Gao et al. (2021) and others selected on the basis of BLAST algorithm-based searches in GenBank (Table 1). A single gene data set was aligned and edited by MAFFT v7.037b (Katoh and Standley 2013) and MEGA v6.0 (Tamura et al. 2013). Combined sequences of ITS, LSU, RPB2 and TEF were performed by SequenceMatrix v.1.7.8 (Vaidya et al. 2011). The model was selected for Bayesian analysis by ModelFinder (Kalyaanamoorthy et al. 2017) in the software PhyloSuite v 1.2.2 (Zhang et al. 2020).

The combined genes were 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 using MrBayes v.3.2 (Ronquist et al. 2012) for the combined sequence datasets. 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 analyses, were discarded, while the remaining 16,001 trees were used for calculating posterior probabilities in the majority rule consensus tree. After the analysis was finished, each run was examined using the program Tracer v1.5 (Drummond and Rambaut 2007) to determine burn-in and confirm that both runs had converged. ML analyses were constructed with IQ-TREE (Trifinopoulos et al. 2016) and the model was the default settings.

Results

Phylogenetic analyses

Phylogenetic trees were generated in analysis 1 (to determine the family placement of the new strains) and analysis 2 (to determine the establishment of the new genera in Clavicipitaceae) (Figs 1 and 2, respectively). 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 outgroups in analysis 1, whereas Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken, Hywel-Jones & Samson (CBS 284.36) and P. lavendulum Perdomo, Dania García, Gené, Cano & Guarro (FMR 10376) were used as the outgroups in analysis 2. The concatenated sequences of analysis 1 and 2 included 77 and 68 taxa, respectively, and consisted of 2,313 (ITS, 604; LSU, 570; RPB2, 576; and TEF, 563) and 2,470 (ITS, 583; LSU, 488; RPB2, 627; and TEF, 772) characters with gaps, respectively.

Figure 1. 

A maximum-likelihood phylogenetic tree of Neoaraneomyces and Pseudometarhizium in the order Hypocreales based on multigene dataset (ITS, LSU, RPB2 and TEF). 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 –37,716.4419, which was obtained from an ML analysis of the dataset (ITS+LSU +RPB2+TEF). The parameters of the rate heterogeneity model used to analyze the dataset were estimated using the following frequencies: A = 0.2282, C = 0.2768, G = 0.2781, T = 0.2169; substitution rates AC = 1.4435, AG = 2.2494, AT = 1.4435, CG = 1.0000, CT = 5.4319 and GT = 1.0000, as well as the gamma distribution shape parameter α = 0.6711. The selected models for BI analysis were GTR+F+I+G4 (ITS, LSU and RPB2), and GTR+F+G4 (TEF). The phylogenetic trees (Fig. 1) constructed using ML and BI analyses were largely congruent and strongly supported in most branches. Each family was clustered into an independent clade. The new strains clustered into an independent clade (Clavicipitaceae) with close relationships to Claviceps, Epichloe (Fr.) Tul. & C. Tul., Cephalosporium Corda, Metapochonia Kepler, S.A. Rehner & Humber, Hypocrella Sacc. and Shimizuomyces Kobayasi.

Analysis 2: The final value of the highest scoring tree was –29,543.7455, which was obtained from the ML analysis of the dataset (ITS+LSU+RPB2+TEF). The parameters of the GTR model used to analyze the dataset were estimated based on the following frequencies: A = 0.2303, C = 0.2800, G = 0.2801, T = 0.2096; substitution rates AC = 1.0000, AG = 3.0029, AT = 1.0000, CG = 1.0000, CT = 7.0264 and GT = 1.0000, as well as the gamma distribution shape parameter α = 0.3934. The selected models for BI analysis were GTR+F+I+G4 (ITS+LSU+TEF) and SYM+G4 (RPB2). The phylogenetic trees (Fig. 2) constructed using ML and BI analyses were largely congruent and strongly supported in most branched. Most genera clustered into independent clades. Strains DY101711 and DY101712 clustered into an independent clade while DY101741, DY101742, DY101801, DY101802, SD05361 and SD05362 clustered into two independent clades with close relationship with Metarhiziopsis D.W. Li, R.S. Cowles & C.R. Vossbrinck.

Figure 2. 

A maximum-likelihood phylogenetic tree of two new genera Neoaraneomyces and Pseudometarhizium and 39 genera in Clavicipitaceae, based on multigene dataset (ITS, LSU, RPB2 and TEF). Statistical support values (≥ 50%/0.5) are shown at the nodes for ML bootstrap support/BI posterior probabilities. The new taxa are in bold.

Taxonomy

Neoaraneomyces W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang, gen. nov.

MycoBank No: 842644

Etymology

Referring to a new genus parasitic on spiders

Type species

Neoaraneomyces araneicola W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang.

Description

Colonies on PDA, white to grey, reverse yellowish. Conidiophores mononematous, usually arising from aerial hyphae, phialides solitary or in groups of two to three. Phialides emerging laterally from hyphae, forming a compact hymenium, abruptly narrowing into a neck. Conidia in chains, one-celled, hyaline, fusiform or ellipsoidal.

Host

Spider (Araneidae)

Habitat

Near roads and located on or under rocks.

Sexual morph

Unknown.

Notes

The genera Akanthomyces, Beauveria, Clonostachys, Cordyceps, Engyodontium de Hoog, Gibellula, Hevansia, Hirsutella, Hymenostilbe, Lecanicillium W. Gams & Zare, Ophiocordyceps Petch, Purpureocillium, and Torrubiella Boud. have been reported as spider-pathogenic fungi in Hypocreales (Shrestha et al. 2019). Gibellula is only found on spiders. Neoaraneomyces differs from Gibellua by its paecilomyces-like conidiogenous structures, phialides which were solitary or in groups of two to four, with fusiform to ellipsoidal conidia.

Neoaraneomyces araneicola W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang, sp. nov.

MycoBank No: 842645
Fig. 3

Type

Duyun City (26°21'27.96"N, 107°22'48.22"E), Qiannan Buyi and Miao Autonomous Prefecture, Guizhou, China. On a dead spider (Araneae), 1 October 2019, Wanhao Chen, GZAC DY10171 (holotype); ex-type living cultures, DY101711.

Figure 3. 

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

Description

Spider host completely covered by white mycelium. Conidiophores mononematous, arising from the lateral hyphae. Colonies on PDA, 3.0–3.2 cm diam. after 14 d at 25 °C, white to pale grey, powdery, consisting of a basal felt, reverse yellowish. Prostrate hyphae smooth, septate, hyaline, 1.4–2.2 μm diam. Erect conidiophores usually arising from aerial hyphae. Phialides single or in groups of two to three, 8.9–23.8 × 1.1–1.6 μm, with a cylindrical to ellipsoidal basal portion, tapering into a short distinct neck. Conidia in chains, hyaline, fusiform to ellipsoidal, one-celled, 2.9–4.4 × 1.3–2.0 μm. Sexual state not observed.

Host

Spider (Araneidae).

Habitat

Near the road, located on or under rocks.

Etymology

Referring to the ability to colonize spiders.

Additional strain examined

Duyun City (26°21'27.96"N, 107°22'48.22"E), Qiannan Buyi and Miao Autonomous Prefecture, Guizhou, China. On a dead spider (Araneae), 1 October 2019, Wanhao Chen, DY101712.

Pseudometarhizium W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang, gen. nov.

MycoBank No: 842641

Etymology

Referring to Metarhizium-like colony.

Type species

Pseudometarhizium araneogenum W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang.

Description

Colonies on PDA, light green, reserve brown to light brown. Conidiophores synnematous or mononematous, erect, scattered. Phialides emerging laterally from synnemata or hyphae, forming a compact hymenium, abruptly narrowing into a helical neck. Conidia, one-celled, fusiform or ellipsoidal.

Host

Spider (Araneae).

Habitat

Near the road, located on or under rocks, or on the underside of leaves of broad-leaved plant species.

Sexual morph

Unknown.

Notes

The light green colonies of Pseudometarhizium are similar to those of Metarhizium species. However, Pseudometarhizium is easily distinguished by the combined datasets (ITS+LSU+RPB2+TEF), and had a close relationship with Metarhiziopsis. Pseudometarhizium can be easily distinguished from Metarhiziopsis by its paecilomyces-like structure and absence of sporodochia.

Pseudometarhizium araneogenum W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang, sp. nov.

MycoBank No: 842642
Fig. 4

Type

Duyun City (26°21'27.96"N, 107°22'48.22"E), Qiannan Buyi and Miao Autonomous Prefecture, Guizhou, China. On a dead spider (Araneae), 1 October 2019, Wanhao Chen, GZAC DY10180 (holotype), ex-type living cultures, DY101801.

Figure 4. 

Pseudometarhizium araneogenum A infected spider B, C culture growing on PDA, B front and C the reverse sides of the colony D–L solitary phialides, or groups of two, conidia in short chains and individual. Scale bars: 10 mm (B, C); 10 μm (D–L).

Description

Spider host completely covered by white mycelium. Conidiophores mononematous, arise from the lateral hyphae. Colonies irregularly 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. Sexual state not observed.

Host

Spider (Araneidae).

Habitat

Near the road, located on or under rocks.

Etymology

Referring to the ability to colonize spiders.

Additional specimen examined

Duyun City (26°21'27.96"N, 107°22'48.22"E) Qiannan Buyi and Miao Autonomous Prefecture, Guizhou, China. On a dead spider (Araneae), 1 October 2019, Wanhao Chen, GZAC DY10174, living cultures, DY101741, DY101742.

Remarks

Pseudometarhizium araneogenum distinguished from P. lepidopterorum, which has longer phialides (21.2–33.7 × 1.1–1.4 μm) and smaller conidia (3.1–4.3 × 1.3–1.5 μm).

Pseudometarhizium lepidopterorum W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang, sp. nov.

MycoBank No: 842643
Fig. 5

Type

Sandu County (25°57'22.21"N, 107°57'54.69"E), Qiannan Buyi and Miao Autonomous Prefecture, Guizhou, China. On a pupa (Lepidoptera), 1 May 2019, Wanhao Chen, GZAC SD0536 (holotype), ex-type living cultures, SD05361.

Figure 5. 

Pseudometarhizium lepidopterorum A infected pupa (Lepidoptera) B, C culture on PDA showing B front and C reverse sides of the colony D–L solitary phialides, or groups of two to three, and conidia in short chains and individual. Scale bars: 10 mm (B, C); 10 μm (D–L).

Description. Host pupa completely covered by white mycelium. Conidiophores arising from lateral hyphae of the synnemata. 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. Sexual state not observed.

Host

Pupa (Lepidoptera).

Habitat

On the underside of leaves of broad-leaved plant species.

Additional strain examined

Sandu County (25°57'22.21"N, 107°57'54.69"E) Qiannan Buyi and Miao Autonomous Prefecture, Guizhou, China. On a pupa (Lepidoptera), 1 May 2019, Wanhao Chen, SD05362.

Etymology

Referring to its insect host, order Lepidoptera.

Remarks

Pseudometarhizium lepidopterorum distinguished from P. araneogenum, which has shorter phialides (8.3–23.3 × 1.3–2.2 μm) and longer conidia (3.4–5.8 × 1.4–1.8 μm).

Discussion

Paecilomyces-like conidiogenous structure is common throughout the Hypocreales (Luangsa-ard et al. 2004) and their presence in the new strains make it impossible to identify them using only morphological characteristics. To determine the family placement of the new strains, a phylogenetic tree was constructed with the combined dataset (ITS+LSU+RPB2+TEF) for 14 families of Hypocreales. The new strains clustered into the Clavicipitaceae clade, confirming that they belonged to this family.

Currently, Clavicipitaceae contains 49 genera (Hyde et al. 2020; Mongkolsamrit et al. 2020; Gao et al. 2021). A phylogenetic analysis was carried out based on the available sequences from 39 of these genera. The new strains clustered into independent clades, suggesting that they belong to new genera in the family Clavicipitaceae. Among the genera without available sequences, Helminthascus Tranzschel and Sphaerocordyceps Kobayasi are spider- and insect-associated teleomorph genera without an asexual state (Hyde et al. 2020). The new strains were easily distinguished from Helminthascus and Sphaerocordyceps by their absence of a teleomorph state and pale green color in the natural state. Thus, the new strains are described as two new genera, based on phylogenetic analysis and morphological characteristics.

The evolutionary dynamics of fungi and their hosts are usually described either through coevolution or host shifts (Vega et al. 2009). In a common ecological niche, shifts to new hosts often occur in accordance with the fungal nutrient requirements. The common ancestor of Hypocreaceae and Clavicipitaceae corresponds to a departure from plant-based nutrition to a model that specializes in animals and fungi (Spatafora et al. 2007). Clavicipitaceous fungi, especially those of the genus Metarhizium, are pathogenic to scale insects, white flies and other insect orders. However, few spider-associated species have been reported. Based on comparison of their evolutional relationships with close relatives, we hypothesize that the new spider-associated genera might have undergone host jumps or transferred their nutritional preferences.

Both mononematous and synnematous conidiophores were reported in natural conditions in the present study. Synnematous entomopathogenic fungi (such as Gibellula spp.) are found on the abaxial leaf surfaces of shrubbery, forest floors and shallow soil layers (Hywel-Jones 1996). These entomopathogenic fungi do not spread by airflow diffusion but employ particular strategies, such as producing synnemata and sticky conidia, to accommodate various arthropod activities and facilitate conidial spread (Abbott 2002). In contrast, strains with mononematous conidiophores occur in more open portions of forests and favor dry conidial dispersal (Chen et al. 2020). Pseudometarhizium lepidopterorum was found on the undersides of leaves of broad-leaved plant species, whereas Neoaraneomyces araneicola and P. araneogenum were found near the road and were located on or under rocks. Thus, we speculate that the presence of synnemata may be the result of convergent evolution to adapt to the ecological environment.

Acknowledgements

This work was funded by National Natural Science Foundation of China (31860002), High-level Innovative Talents Training Object in Guizhou Province (Qiankehepingtairencai [2020]6005), Science and Technology Foundation of Guizhou Province (Qiankehejichu [2020]1Y060), Program of Innovative Scientific and technological Talent Team of Guizhou Province (2020-5010), Construction Program of Guizhou Engineering Research Center (Qian Fa Gai Gao Ji 2020-896), Guizhou Science and Technology Support Project (Qiankehezhicheng [2019]2776).

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Supplementary material

Supplementary material 1 

Table S1

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

Data type: COL.

Explanation note: Primers information for 5 DNA sequences.

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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