Comprehensive Review of Tolypocladium and Description of a Novel Lineage from Southwest China

Abstract

Tolypocladium, a diverse genus of fungicolous fungi belonging to Ophiocordycipitaceae, includes saprotrophic soil inhabitants, plant endophytes and pathogens of insects, nematodes, rotifers, and parasites of truffle-like fungi. Here, we review the research progress achieved for Tolypocladium regarding its taxonomy, species diversity, geographic distribution, host affiliations and ecological diversity. Furthermore, an undescribed taxon from China was established using morphology and multi-gene phylogeny. Tolypocladium inusitaticapitatum is introduced as a new species parasitizing ectomycorrhizal Elaphomyces species. It is diagnosed by its irregularly enlarged fertile heads and lemon, yellow-to-dark-brown, smooth and nearly cylindrical stipe. Phylogenetic analyses based on SSU, LSU, ITS, TEF1-α and RPB2 sequence data showed T. inusitaticapitatum to be an independent lineage separated from T. flavonigrum in the clade comprising T. capitatum, T. fractum and T. longisegmentatum. A key for identifying the sexual Tolypocladium species is also provided.

Full text

pathogens
Article
Comprehensive Review of Tolypocladium and Description of a
Novel Lineage from Southwest China
Feng-Ming Yu 1,2,3, Kandawatte Wedaralalage Thilini Chethana 1,2, De-Ping Wei 1,2, Jian-Wei Liu 1,2,4,
Qi Zhao 3,5,6,7, Song-Ming Tang 1,2,8, Lu Li 3,5 and Kevin David Hyde 1,2,3,*


Citation: Yu, F.-M.; Thilini Chethana,
K.W.; Wei, D.-P.; Liu, J.-W.; Zhao, Q.;
Tang, S.-M.; Li, L.; Hyde, K.D.
Comprehensive Review of
Tolypocladium and Description of a
Novel Lineage from Southwest China.
Pathogens 2021,10, 1389. https://
doi.org/10.3390/pathogens10111389
Academic Editor: LászlóKredics
Received: 14 September 2021
Accepted: 20 October 2021
Published: 27 October 2021
Publisher’s Note: MDPI stays neutral
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand;
6171105508@lamduan.mfu.ac.th (F.-M.Y.); kandawatte.thi@mfu.ac.th (K.W.T.C.);
wei_deping@cmu.ac.th (D.-P.W.); liujianwei@mail.kib.ac.cn (J.-W.L.);
6171105516@lamduan.mfu.ac.th (S.-M.T.)
2School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
3Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming 650201, China; zhaoqi@mail.kib.ac.cn (Q.Z.);
lilu@mail.kib.ac.cn (L.L.)
4
The Germplasm Bank of Wild Species, Yunnan Key Laboratory for Fungal Diversity and Green Development,
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China
5Yunnan Key Laboratory for Fungal Diversity and Green Development, Kunming 650201, China
6Institute of Applied Fungi, Southwest Forestry University, Kunming 650224, China
7School of Chemical Engineering, Guizhou Institute of Technology, Guiyang 550003, China
8Biotechnology and Germplasm Resources Institute, Yunnan Academy of Agricultural Science,
Kunming 650205, China
*Correspondence: kdhyde3@gmail.com
Abstract:
Tolypocladium, a diverse genus of fungicolous fungi belonging to Ophiocordycipitaceae,
includes saprotrophic soil inhabitants, plant endophytes and pathogens of insects, nematodes,
rotifers, and parasites of truffle-like fungi. Here, we review the research progress achieved for
Tolypocladium regarding its taxonomy, species diversity, geographic distribution, host affiliations
and ecological diversity. Furthermore, an undescribed taxon from China was established using
morphology and multi-gene phylogeny. Tolypocladium inusitaticapitatum is introduced as a new
species parasitizing ectomycorrhizal Elaphomyces species. It is diagnosed by its irregularly enlarged
fertile heads and lemon, yellow-to-dark-brown, smooth and nearly cylindrical stipe. Phylogenetic
analyses based on SSU, LSU, ITS, TEF1-
α
and RPB2 sequence data showed T. inusitaticapitatum to be
an independent lineage separated from T. flavonigrum in the clade comprising T. capitatum,T. fractum
and T. longisegmentatum. A key for identifying the sexual Tolypocladium species is also provided.
Keywords: new taxon; diversity; ecology; host shift; multi-gene; mycoparasite; taxonomic key
1. Introduction
Fungal species establish antagonistic to mutualistic associations with numerous
prokaryotes and eukaryotes, including bacteria, algae, animals, plants and other fungi [
1
].
More than 1500 fungicolous taxa are widely distributed in aquatic and terrestrial ecosys-
tems from tropical to polar regions [
1
]. Their hosts are ecologically diverse across the fungal
kingdom. Truffle-like fungi are hypogeous and taxonomically distributed in Ascomycota
and Basidiomycota [
2
]. Some truffle-like fungi were reported to be hosts of fungicolous
species belonging to Absidia Tiegh., Battarrina (Sacc.) Clem. and Shear, Entoloma P. Kumm.,
Hypocrea Fr., Hypomyces (Fr.) Tul. and C. Tul., Hypoxylon Bull., Melanospora Corda, Sporothrix
Hektoen and C.F. Perkins, and Tolypocladium W. Gam [1,3].
Tolypocladium W. Gams was established based on three soil-inhabiting asexual species:
Tolypocladium cylindrosporum W. Gams, T. geodes W. Gams and T. inflatum W. Gams (the
type species) [
4
]. Hodge and colleagues linked the asexual T. inflatum to the sexual species
Pathogens 2021,10, 1389. https://doi.org/10.3390/pathogens10111389 https://www.mdpi.com/journal/pathogens

Pathogens 2021,10, 1389 2 of 14
Cordyceps subsessilis Petch [
5
]. Subsequently, Sung and colleagues introduced the sexual
genus Elaphocordyceps G.H. Sung and Spatafora and linked it to the asexual Tolypocladium
and some species within Verticillium Nees based on multigene phylogeny [
6
]. Moreover,
Sung and colleagues transferred the species of Cordyceps sensu lato that parasitize ectomyc-
orrhizal Elaphomyces (18 species and two forma), cicada nymphs (C. inegoënsis Kobayasi,
C. paradoxa Kobayasi, and C. toriharamontana Kobayasi) and beetle larvae (C. subsessilis) to
Elaphocordyceps [
6
]. Chaunopycnis was established by Gams to accommodate Ch. alba, which
resembles Tolypocladium in conidiogenesis [
7
]. Later, Quandt and colleagues synonymized
Chaunopycnis and Elaphocordyceps under Tolypocladium, following the “One Fungus One
Name” rule, as Tolypocladium is much more widely known, medicinally important and an
older genus [4,6–8].
Most Tolypocladium species are Elaphomyces-attacking mycoparasites, except for few
entomopathogens [
9
,
10
]. The evolution of host specificity and the dynamics of host jumping
were investigated by several researchers using molecular data [
6
,
8
,
11
–
15
]. Nikoh and
Fukatsu inferred that there was a shift from entomoparasitism to mycoparasitism during
the evolution of the Cordyceps-like fungi [
11
]. However, with the addition of more gene
regions and taxa, insect pathogens such as T. paradoxum and T. inflatum were found to
be clustered with some parasites on truffles. The researchers explained that the ancestral
ecology was a truffle parasitism, with multiple switches to insect pathogenicity [
6
,
8
,
12
].
Notably, the interspecific relationships of closely related Tolypocladium species are weakly
supported and inconsistently resolved with different datasets [
6
,
8
,
13
,
14
]. To compensate for
the shortage of limited loci, Quandt and colleagues performed genome-scale phylogenetic
analyses based on two entomopathogens (T. ophioglossoides and T. capitatum) and two
mycoparasites (T. inflatum and T. paradoxum) and demonstrated that truffle parasites form
a monophyletic clade. They suggest that this lineage is derived as a result of a single
ecological transition or host-jumping from insects to fungi [15].
A successful infection caused by fungal pathogens generally undergoes host recog-
nition, attachment, and then infection and degradation, depending on the gene content,
expression, or regulation [
16
]. Tolypocladium is recognized as an ideal candidate for inves-
tigating the mechanisms associated with host-jumping [
15
,
16
]. Quandt and colleagues
researched the set of genes that are differentially regulated in Tolypocladium species dur-
ing their first encounter with their hosts [
16
]. They found that PTH11-related G-protein-
coupled receptors (GPCRs), predicted to be involved in host recognition, were up-regulated
in
T. ophioglossoides
when grown on media containing insect cuticles [
16
]. Furthermore, a
divergent chitinase and an adhesin gene, Mad1, were significantly up-regulated on me-
dia containing Elaphomyces [
16
]. According to the transcriptomic data, genes involved
in redox reactions and transmembrane transport were the most overrepresented during
T. ophioglossoides
growth on Elaphomyces media. However, the genes involved in secondary
metabolism may not be necessary for the parasitism of truffles as their products are only
highly expressed during the growth on insect tissues [16].
To date, Tolypocladium comprises 41 species (Table 1) with a cosmopolitan distribu-
tion [
2
,
17
]. Some of them produce various secondary metabolites, such as cyclosporin,
efrapeptins, ophiocordin and ophiosetin [
18
]. They have been widely used in biophar-
maceuticals and biocontrol [
18
]. During an investigation of fungi in Yunnan Province,
Southwest China, an undescribed Tolypocladium species was discovered on Elaphomyces
sp. The present study aimed to (i) systematically review species diversity, hosts/habitat,
geographical distribution and host affiliations of Tolypocladium species, (ii) broaden the
knowledge of species diversity and host shifts in Tolypocladium species, (iii) refine the
diagnostic characters of the interspecific classification of Tolypocladium in sexual morphs
and provide a taxonomic key.

Pathogens 2021,10, 1389 3 of 14
Table 1. Species diversity, hosts/habitats and geographic distribution of Tolypocladium species.
Fungal Name Hosts/Isolated From Known Distribution
T. album Soil, sapwood of Hevea brasiliensis Colombia, France, Scotland, Sri Lanka, Sweden,
The Netherlands [7], Peru [12]
T. amazonense Sapwood of Hevea brasiliensis and H. guianensis Peru [12]
* T. capitatum Elaphomyces granulatus,E. japonicus,Elaphomyces sp. Asia (China (Taiwan, Yunnan), Japan), Europe (France, Holland,
Hungary), North America (Canada, U.S.A.) [9,10,19–22]
T. cylindrosporum
Soil, sewage, peat, roots of Picea mariana;Plecia nearctica,
larvae of Aedes sierrensis, larvae of Aedes australis, larvae
and pupae of Lucilia sericata,Drosophila larvae (Diptera)
Brazil, China, Czech, England, New Zealand, Nepal, The
Netherlands, The North Island, U.S.A. [4,23–27]
* T. delicatistipitatum E. asahimontanus China (Jiangxi) [28], Japan [10]
* T. dujiaolongae Cicada nymphs China (Anhui, Fujian, Jiangsu, Jiangxi, Zhejiang) [29]
T. endophyticum Living sapwood of Hevea brasiliensis and H. guianensis Brazil, Mexico, Peru [12]
T. extinguens Larvae of Arachnocampa luminosa (Diptera) New Zealand [24]
* T. fractum E. appalachiensis U.S.A. (Tennessee) [9]
* T. flavonigrum Elaphomyces sp. Thailand [30]
* T. fumosum Cocooned pupa of bagworm moth (Psychidae) buried
among mosses Poland [31]
T. geodes Soil Austria, Canada, China, Denmark, England,
The Netherlands [4,23,26]
* T. guangdongense Elaphomyces sp. China (Guangdong) [32]
* T. inegoense Cicada nymphs (e.g., Hyalessa maculaticollis) China (Fujian, Taiwan) [33], Japan [34], Korea [6]
* T. inflatum
Larvae of Scarabaeidae (e.g., Aphodiinae,Rutelinae) (sexual
morph); soil, humus, Picea glauca, roots of P. mariana,
surface of Mycobates sp. (Acari,Mycobatidae), sclerotium of
Ophiocordyceps gracilis (asexual morph)
Sexual morph: Japan, U.S.A. (Tennessee, North Carolina, Michigan,
New York, Washington) [5]; asexual morph: Austria, Canada, China,
Nepal, Germany, U.S.A. [4,23,26,35]
* T. intermedium E. granulatus,E. subvariegatus Japan, U.S.A. (New York) [10,36]
* T. japonicum E. granulatus,E. japonicus,E. neoasperulus Austria, Japan [10], China (Guizhou, Taiwan) [28,37]
* T. jezoense E. anthracinus,E. miyabeanus,E. nopporensis Japan [10]
T. lignicola Rotting wood (parasitic in bdelloid rotifers) Canada (Ontario) [38]
* T. longisegmentatum E. granulatus,E. japonicus,E. muricatus,Elaphomyces sp. Asia (China (Jilin), Japan), Europe (England, Germany, Holland),
North America (Canada, Mexico, U.S.A.) [9,10,20,21,39]
T. microsporum Soil Canada, Germany, The Netherlands, U.S.A. [23]
* T. minazukiense Elaphomyces sp. Japan [40]
* T. miomoteanum Elaphomyces sp. Japan [40]
T. nubicola Soil Canada (Alberta), China (Guizhou) [23,41]
* T. ophioglossoides E. granulatus,E. japonicus,E. muricatus,E. shimizuensis,E.
titibuensis, and Elaphomyces sp.
Commonly in Asia (e.g., China (Guangxi, Jiangsu, Jiangxi, Jilin,
Shandong, Sichuan, Taiwan, Yunnan), Japan, Korea), Europe and
North America [9,10,42–44]
T. ovalisporum Lichen Polycauliona regalis Antarctica (King George Island) [45]
* T. paradoxum Cicada nymphs (e.g., Platypleura kaempferi,
Graptopsaltria nigrofuscata)China (Hainan, Yunnan) [46], Japan, Koera [34,47]
T. pustulatum Soil, twigs in oak forest, and living leaf of Kalmia latifolia Mexico (Nuevo León), Spain (Cádiz), U.S.A. (New Jersey) [48]
* T. ramosum Elaphomyces sp. China (Anhui, Fujian, Gansu, Guangdong) [44,49,50]
* T. rouxii E. variegatus France [51]
T. sinense Stroma and sclerotium of Ophiocordyceps sinensis China (Yunnan) [52]
* T. szemaoense E. granulatus China (Yunnan) [53]
* T. tenuisporum Host not found (probably Elaphomyces sp.) U.S.A. (Pennsylvania) [9]
T. terricola Soil Finland [54]
* T. toriharamontanum Cicada nymph (Auritibicen bihamatus) Japan [34]
T. trigonosporum Rotting stump (parasitic on bdelloid rotifers) Canada (Nova Scotia) [55]
T. tropicale Sapwood and leaf tissue of Hevea brasiliensis Mexico, Peru [12]
T. tundrense Soil Canada (Northwest Territories) [23]
* T. valliforme E. granulatus,Elaphomyces sp. Canada (Ontario), U.S.A. (Carolina, New York, Virginia) [9]
* T. valvatistipitatum E. granulatus,E. neoasperulus Japan [10]
* T. virens Elaphomyces sp. Japan [56]
* indicates sexual morphs (25 species).

Pathogens 2021,10, 1389 4 of 14
2. Results
2.1. Phylogenetic Placement
The combined SSU, LSU, ITS, TEF1-
α
and RPB2 sequence dataset comprised 35 species,
containing 5384 nt (SSU: 1–1536, LSU: 1537–2441, ITS: 2442–3306, TEF1-
α
: 3307–4264, RPB2:
4265–5384) after the alignment (including gaps). Among them, 3731 bp (base pairs) were
conserved, 378 variable, parsimony-uninformative, and 1275 parsimony-informative. The
ML and BI analyses resulted in phylogenetic trees with a similar topology. The ML tree with
a final log-likelihood of
−
27186.604 is shown in Figure 1. Specimens HKAS 112152 and
HKAS 112153 clustered together and formed a distinct clade with strong support values
(SH-aLRT = 100, UFB = 100 and BIPP = 1), indicating a conspecific relationship. These two
specimens separated from other Tolypocladium species with SH-aLRT = 90.2 and
BIPP = 0.98
support values. However, their LSU sequences showed an 11 bp difference (1.28%) across
the 862 bp region, contributing to the different branch lengths in the phylogenetic tree.
Based on the available molecular data for Tolypocladium species, some differences are known
to occur due to intraspecific variations in the LSU sequences, ranging from 0.25 to 1.28%
(Table 2).
Table 2. Intraspecific base-pair differences in LSU genes among Tolypocladium species.
Species
Locus 522 532 855 Ratio
T. album CBS 393.89 #CCC 0.35% (3/870 bp)
GB5502 T T -
Species
Locus 20 21 23 24 25 27 Ratio
T. inflatum OSC 71235 #A G A A C A 0.76% (6/794 bp)
CBS 127302 G A - - - C
Species
Locus 48 434 Ratio
T. ophioglossoides CBS 100239 #C C 0.25% (2/816 bp)
NBRC 106330 T T
Species
Locus 164 382 405 433 442 479 496 524 Ratio
T. paradoxum NBRC 106958 #T C G C C C T G 0.90% (8/891 bp)
NBRC 100945 C T A T T T C A
Species
Locus 8 37 44 51 81 96 110 124 204 210 402 Ratio
T. inusitaticapitatum HKAS 112152 #T T A A A A T T A A G 1.28% (11/862 bp)
HKAS 112153 C C G G G G C C G G T
The locus numbers refer to the base-pair positions of the gene sequences, and “
#
” represents the reference sequences. Gaps are indicated
with ‘-’.
Specimens Tolypocladium inusitaticapitatum (China), together with four Tolypocladium
species occurring on Elaphomyces spp., i.e.,
T. capitatum
(intercontinental distribution),
T. flavonigrum
(Thailand), T. fractum (USA) and T. longisegmentatum (intercontinental dis-
tribution), formed a monophyletic clade with weak support (SH-aLRT = 81.1, UFB = 82
and BIPP = 0.90. UFB values not shown in the ML tree). Tolypocladium inusitaticapitatum
formed a separate clade sister to T. flavonigrum. However, the nucleotide comparison
between T. inusitaticapitatum (holotype: HKAS 112152) and T. flavonigrum (holotype: BCC
66576) showed 154 bp (26.78%) differences across 575 bp ITS, 87 bp (9.83%) differences
across
885 bp
LSU, and 47 bp (4.99%) differences across 942 bp TEF1-
α
(including gaps),
respectively. The phylogenetic evidence suggested that these two specimens represent
new species.
2.2. Taxonomy
Tolypocladium W. Gams, Persoonia 6: 185 (1971); emended by Quandt and colleagues,
IMA Fungus 5: 125 (2014).
Index Fungorum number: IF10242; Facesoffungi number: FoF 10425.
Synonyms:Chaunopycnis W. Gams, Persoonia 11: 75 (1980).
Elaphocordyceps G.H. Sung and Spatafora, Stud. Mycol. 57: 36 (2007).
Type species:Tolypocladium inflatum W. Gams 1971.

Pathogens 2021,10, 1389 5 of 14
Pathogens 2021, 10, x FOR PEER REVIEW 6 of 17
Figure 1. Maximum likelihood (ML) tree of Tolypocladium inusitaticapitatum and its allies within Ophiocordycipitaceae in-
ferred from combined SSU, LSU, ITS, TEF1-α and RPB2 dataset. Bootstrap support values for ML ≥ 80 of SH-aLRT or 95
of UFB and posterior probability for BI ≥ 0.90 are indicated above the nodes and separated by ‘-/-/-’ (SH-aLRT/UFB/BIPP).
Specimens of the current study are given in red. Type specimens are in bold and the superscript ‘ex’ indicates ex-type.
2.2. Taxonomy
Tolypocladium W. Gams, Persoonia 6: 185 (1971); emended by Quandt and colleagues,
IMA Fungus 5: 125 (2014)
Index Fungorum number: IF10242; Facesoffungi number: FoF 10425
Synonyms: Chaunopycnis W. Gams, Persoonia 11: 75 (1980)
Figure 1.
Maximum likelihood (ML) tree of Tolypocladium inusitaticapitatum and its allies within Ophiocordycipitaceae inferred
from combined SSU, LSU, ITS, TEF1-
α
and RPB2 dataset. Bootstrap support values for ML
≥
80 of SH-aLRT or 95 of
UFB and posterior probability for BI
≥
0.90 are indicated above the nodes and separated by ‘-/-/-’ (SH-aLRT/UFB/BIPP).
Specimens of the current study are given in red. Type specimens are in bold and the superscript ‘ex’ indicates ex-type.
Morphological characterization:Sexual morph:Stromata arise directly from the host
and are sometimes indirectly connected to the host through rhizomorph-like structures.
They range from solitary to several and can be simple or branched. Stipe is fibrous to
tough, rarely fleshy, dark-brownish to greenish with an olivaceous tint, rarely whitish,
cylindrical and enlarges near the fertile part. The fertile part is clavate- to capitate-shaped

Pathogens 2021,10, 1389 6 of 14
and varies in color. Perithecia are partially to completely immersed, or superficial, or
produced on a highly reduced stromatic pad, and ostiolate. Asci are unitunicate and
long cylindrical with a thickened apical cap. Ascospores are filiform, approximately as
long as asci, multi-septate, typically disarticulate into part-spores, and are occasionally
non-disarticulating when mature (e.g., T. ramosum). Part-spores are hyaline, fusiform to
cylindrical with round to truncate ends [
6
,
8
]. Asexual morph: They are Tolypocladium-,
Chaunopycnis-, or Verticillium-like. Colonies are white, cottony and grow slowly on artificial
media (e.g., potato dextrose agar, Czapek
–
Dox agar, malt extract agar, Sabouraud Glucose
agar and water agar). Conidiophores usually are short and bear lateral or terminal phialides
whorls. Phialides usually are swollen at the base and thin, often with bent necks. Conidia
are globose to oval, one-celled, hyaline, smooth, and aggregative in small heads at the tips
of the phialides [4,23].
Hosts and habits: Found in terrestrial and humid environments. Species of Tolypocladium
parasitize hypogeous Elaphomyces (20 species including the novel species described in this
study), cicada nymphs (4 species), beetle larvae (T. inflatum), pupa of the bagworm moth
(T. fumosum), mosquito larvae (T. extinguens), and even bdelloid rotifers exposed to air
(
T. lignicola
and T. trigonosporum). Their ascospores/conidia and mycelia survive in soil, or
on various humus, rotting wood, plant tissues and surfaces, body surfaces of insects and
mites, tissues of Cordyceps and lichens (Table 1).
Species diversity and distribution:Tolypocladium currently consists of 42 species (includ-
ing the novel species described in this study) distributed worldwide [
2
,
3
,
17
]. Fifteen species
were recorded from China (Table 1).
2.3. Description of the Novel Species
Tolypocladium inusitaticapitatum F.M. Yu, Q. Zhao and K.D. Hyde, sp. nov. Figure 2.
Index Fungorum: IF558123; Facesoffungi number: FoF 10407.
Typification: China, Yunnan Province, Lijiang City, Lijiang Alpine Botanic Garden,
E100
◦
10
0
58.07, N26
◦
59
0
58.35, alt. 3338 m, 5 Oct 2019, Jian-Wei Liu (HKAS 112152, holotype).
Etymology: The specific epithet ‘inusitaticapitatum’ is derived from the combination
of two Latin words, 1) adjective inusitata (strange, odd) and 2) noun capitatum (head),
pointing to the fertile head, which is irregularly expanded.
GenBank accession numbers: ITS = MW 537735, LSU = MW 537718, SSU = MW 537733,
TEF1-α= MW 507527, RPB2 = MW 507529.
Description: Asexual morph Stromata 9–11.5 cm high, solitary and simple, arising di-
rectly from the fruiting bodies of Elaphomyces sp. Stipe yellow at base, olive-brown to dark
brown at the middle part, and yellowish brown at the terminal part. They are 7.5–11.5 cm
long and 7–8.5 mm thick in the widest parts and nearly cylindrical, but the middle part is
slightly thicker than the basal and upper parts. The fertile part developed from the terminal
of the stipe, and is somewhat ellipsoidal, irregularly barrel-shaped, and sometimes slightly
compressed, 1.5–2.0 cm
×
1.5–2.0 cm. The surface is decorated with white ascospores
released from the mature perithecia, which is olive yellow when immature, and olive to
dark brown when mature. The outer layer becomes cracked and the olive internal texture
is exposed. Structure of cortex of fertile part: composed of olive brown pseudoparenchyma-
tous tissue and an ectal layer. Perithecia
580–720 µm×180–270 µm
(
x = 650 µm×220 µm,
n= 10
), crowded, entirely immersed, obovoid, ellipsoidal to pyriform. Ostioles papillate,
and are visible (protruding up to 55
µ
m in high) or invisible, lined with periphyses. Asci
is 410–510
µ
m
×
10–15
µ
m (x = 461
µ
m
×
13
µ
m, n= 20), hyaline, and long cylindrical,
with a conspicuously thickened cap (measuring 6.5–7.5
µ
m
×
6.0–7.0
µ
m). Ascospores are
approximately as long as asci, and extremely easy to break into part-spores. Part-spores
20–32
µ
m
×
3.0–4.5
µ
m (x = 25
µ
m
×
3.6
µ
m, n= 20), hyaline, cylindrical with rounded
ends. Asexual morph: Unknown.

Pathogens 2021,10, 1389 7 of 14
Pathogens 2021, 10, x FOR PEER REVIEW 9 of 17
Figure 2. Tolypocladium inusitaticapitatum (holotype: HKAS 112152). (a) Habitat; (b) Stromata arising from the fruiting bod-
ies of Elaphomyces sp.; (c) Fertile heads; (d) Decomposed Elaphomyces sp.; (e) Ascospores released from mature perithecia
(shown by a red arrow); (f) Vertical section of a fertile head; (g) Median section across the ostiole of the perithecium; (h)
Vertical section across the cortex of a fertile head; (i–n) Asci with ascospores; (o) A thickened cap; (p,q) Part-spores. Bars:
(b) = 10 cm; (c,d) = 2 cm; (e) = 2 mm; (f) = 500 μm; (g) = 50 μm; (h) = 100 μm; (i–n) = 250 μm; (o–q) = 20 μm.
Notes: Based on the multi-gene phylogeny results, our specimens are closely related
to Tolypocladium flavonigrum, known only from Thailand. Both species have stromata di-
rectly emerging from the surface of Elaphomyces sp., and capitate fertile heads with the
perithecia entirely immersed in a well-differentiated valliforme-like structure [30]. How-
ever, T. inusitaticapitatum considerably differs from T. flavonigrum for the olive, yellowish-
brown to dark brown fertile part, and is yellow to yellowish-brown at both ends of the
stipe compared to the yellow–black to black stromata in T. flavonigrum. Tolypocladium
Figure 2.
Tolypocladium inusitaticapitatum (holotype: HKAS 112152). (
a
) Habitat; (
b
) Stromata arising from the fruiting
bodies of Elaphomyces sp.; (
c
) Fertile heads; (
d
) Decomposed Elaphomyces sp.; (
e
) Ascospores released from mature perithecia
(shown by a red arrow); (
f
) Vertical section of a fertile head; (
g
) Median section across the ostiole of the perithecium;
(h) Vertical
section across the cortex of a fertile head; (
i
–
n
) Asci with ascospores; (
o
) A thickened cap; (
p
,
q
) Part-spores. Bars:
(b) = 10 cm; (c,d)=2cm;(e)=2mm;(f) = 500 µm; (g) = 50 µm; (h) = 100 µm; (i–n) = 250 µm; (o–q) = 20 µm.
Host and habitat: Directly arising from the fruiting bodies of hypogeous Elaphomyces sp.
(Elaphomycetaceae,Eurotiales), in a humid and evergreen broad-leaved rainforest (Lijiang
Alpine Botanic Garden), Lijiang, Yunnan Province, P.R. China. As serious degradation
has occurred, truffle-like Elaphomyces sp. could not show any morphological evidence
of taxonomic significance. Based on the ITS sequence dataset, the phylogenetic analyses
showed that the host of T. inusitaticapitatum clustered together with Elaphomyces fuscus
M. Shirakawa (Japan) and formed a sister group. However, there are sufficient molecular
differences between the host from HKAS 112152 (ITS = MW 513695) and E. fuscus F-a170629
(ITS = LC 500967) to consider them as distinct species.
Known distribution: P.R. China (Yunnan).
Other specimen examined: CHINA, Yunnan, Lijiang, Lijiang Alpine Botanic Garden, alt.
3338 m, 5 October 2019, Jian-Wei Liu (HKAS 112153).

Pathogens 2021,10, 1389 8 of 14
Notes: Based on the multi-gene phylogeny results, our specimens are closely related
to Tolypocladium flavonigrum, known only from Thailand. Both species have stromata di-
rectly emerging from the surface of Elaphomyces sp., and capitate fertile heads with the
perithecia entirely immersed in a well-differentiated valliforme-like structure [
30
]. How-
ever,
T. inusitaticapitatum
considerably differs from T. flavonigrum for the olive, yellowish-
brown to dark brown fertile part, and is yellow to yellowish-brown at both ends of the
stipe compared to the yellow
–
black to black stromata in T. flavonigrum.Tolypocladium
inusitaticapitatum produces obovoid, ellipsoidal to pyriform perithecia, which are markedly
distinguished from the elongate-ovoid perithecia produced by T. flavonigrum. Asci and part-
spores of T. inusitaticapitatum (410–510
µ
m
×
10–15
µ
m, 20–32
µ
m
×
3.0–4.5
µ
m) are larger
than those of T. flavonigrum ((318–)330–416(–482) µm×7–8 µm, 2–5 µm×1.5–2 µm) [30].
When comparing Tolypocladium inusitaticapitatum with its other phylogenetic relatives
(
T. capitatum
,T. fractum and T. longisegmentatum), differences were found. Tolypocladium capitatum
differs from T. inusitaticapitatum mainly due to its larger perithecia (
900–1100 µm×340–430 µm
)
and slimmer part-spores (2.5–3
µ
m wide) [
10
]. Tolypocladium fractum differs from T. inusitati-
capitatum by having smaller stromata (1.5–2.5 cm long) and asci (
300–480 µm×5–6 µm
) [
10
].
Tolypocladium longisegmentatum is distinguished from T. inusitaticapitatum by its longer
stipe (13 cm long when fresh and up to 11 cm long when dried) and longer part-spores
((12–)40–65 µm)
[
20
]. Morphologically, T. inusitaticapitatum is similar to T. intermedium for
the yellow to dark brown stipe but differs in its smaller asci and shorter part-spores (main
differences are outlined in Table 3). Regretfully, the molecular data of T. intermedium is not
available in GenBank.
Table 3. Main differences between T. intermedium and T. inusitaticapitatum.
T. intermedium [10]T. inusitaticapitatum (This Study)
Fertile part Dark reddish brown Olive brown, yellowish-brown to dark brown
Stipe
Slender, 6–8.5 cm long and 2–4 mm thick, middle
part clearly expanded, surface with many
longitudinal grooves, upper part squamulose
Thicker, 7.5–11.5 cm long and 7–8.5 mm thick, middle
part indistinctly expanded, surface smooth
Asci 240–300 µm×7–8 µm, caps about 5 µm
in diameter 410–510 µm×10–15 µm, caps 6.5–7.5 µm×6.2–7.0 µm
Part-spores Short, 3–6 (commonly 4.5) µm×1.5–2 µm,
truncated at two ends (shape)
Long, 20–32 µm×3.0–4.5 µm, cylindrical with
rounded ends
Distribution Japan, USA P.R. China (Yunnan)
3. Discussion
Tolypocladium, a generalist genus, has been reported to have diverse lifestyles on a
wide range of hosts and environments, including soil, insects, plants, lichens and hypogeal
fungi [
6
,
8
]. The current pattern of host affiliation of Tolypocladium fungi is inferred to
be an evolutionary product of intra- and inter-kingdom host shifts [
57
]. In the last two
decades, researchers aimed to infer the evolution of host affiliation within the Tolypocladium,
either using a handful of gene loci from dozens to hundreds of taxa, or using genome-
scale data from fewer taxa [
11
,
12
,
15
,
58
]. To date, the studies on the host-jumping of
Tolypocladium have been performed with multigene phylogeny (seven genes from 202 taxa of
Hypocreales) [
12
] and genome-scale phylogeny (1350 genes from 20 taxa of Hypocreales) [
15
].
The multigene phylogenies supported three hypotheses for Tolypocladium, as follows:
(1) the
ancestral hosts were fungi (false truffles) [
11
,
12
,
57
,
58
]; (2) there were multiple switches
to insect pathogenesis from a mycoparasitic ancestor [
8
,
12
,
13
]; (3) the endophytic lineage
has arisen with the contact of plant hosts via mycorrhizal associations or plant-associated
insects [
12
]. However, these conclusions, made from multigene phylogenies, conflict with
those made from genome-scale phylogenies, which suggested a single ecological transition
from insects to fungi within Tolypocladium [
15
]. Our phylogenic tree, inferred from five
genes of 35 species (Figure 1), resulted in consistent conclusions, similar to those from

Pathogens 2021,10, 1389 9 of 14
previous multigene phylogenies. Similarly, we encountered several problems, such as
phylogenetic conflicts among genetic data partitions and moderate to low support values
for some important nodes [
8
,
12
,
13
]. Although whole-genome data provide insights that can
further resolve the phylogenetic relationships of Tolypocladium [
15
,
59
,
60
], it is still unknown
whether those conclusions will be limited by the few available species.
In this study, a novel Tolypocladium species occurring on Elaphomyces sp. is known from
its sexual morph. A taxonomic key is also provided for 26 Tolypocladium species. The shape
of the fertile part, the connection between the stipe and host, the structure of the cortex of
the fertile part, size of part-spores and host affiliation are thought to be characteristic of
taxonomic significance for interspecific identification [
8
–
10
]. However, there are
16 species
whose sexual morphs are still unknown. In addition, the phylogenetic relationships among
Tolypocladium species are very sensitive to taxa sampling and loci information [
8
,
15
]. Further
studies should focus on obtaining more samples from different geographic regions and/or
ecological niches, sequencing more markers and even genomic data, building a more
robust phylogenetic relationship, and establishing their sexual
–
asexual morph connections.
(Table 4).
Table 4. Key to Sexual Morphs of Tolypocladium species.
1. Host insects 2
10. Host hypogeous Elaphomyces spp. 7
2. Host beetle or moth larvae 3
20. Host cicada nymphs 4
3. Fertile part capitate, with stellate appearance; perithecia ovoid to pear-shaped, 740–760 ×444–558 µmT. fumosum
30. Fertile part, strap-shaped pseudostalk; perithecia superficial, narrow flask-shaped, 1000–1500 ×330–440 µmT. inflatum
4. Stromata arising from underground mycelial membrane or strand; part-spores 3–5 ×1.5–2 µmT. paradoxum
40. Stromata arising directly from host 5
5. Fertile part elongated, obpyriform; part-spores 1.5–2–2.5 ×1.5–1.7 µm wide T. toriharamontanum
50. Fertile part oblong or clavate 6
6. Perithecia superficial or apparently half-immersed, pyriform, 520–550 ×260–280 µm; part-spores 2.5–3 ×2µmT. inegoense
60. Perithecia wholly immersed, ampullaceous, (233–)520–740(–780) ×(250–)300–330 (–360) µm; part-spores 3–5(–7.0) ×2–3 µmT. dujiaolongae
7. Stroma attached to host by rhizomorphs 8
70. Stroma arising directly from the host 12
8. Part-spores articulate, moniliform, 3–3.5 ×2–2.5 µmT. szemaoense
80. Part-spores with truncate or rounded ends 9
9. Stroma capitate 10
90. Stroma solitary or rarely caespitose 11
10. Perithecia small, 480–540 ×225–255 µm; part-spores large-sized, 18–28 ×3–5 µmT. delicatistipitatum
100. Perithecia 770–800 ×350–430 µm; part-spores medium-sized, 8–11 ×1.5–2 µmT. miomoteanum
11. Perithecia oblong with long neck, 700–720 ×200–250 µm; part-spores long, 20–30(50) ×3–4.5 µmT. jezoense
110. Perithecia ovoid, 550–600 ×200–300 µm; part-spores small short rod-shaped, 2.5–5 ×1.5–2 µmT. ophioglossoides
12. Perithecia superficial, ascospores nonfractured T. ramosum
120. Perithecia entirely embedded or ostiole slightly projecting 13
13. Fertile part, cortex composed of pseudoparenchymatous peridial layer, and with an ectal layer 14
130. Fertile part, cortex composed of pseudoparenchymatous peridial layer, but without ectal layer 19
14. Stromata clavate; perithecia narrowly ovoid, 750–1000 ×250–300 µm; part-spores cylindric, 6–8 ×1–1.5 µmT. tenuisporum
140. Stromata capitate 15
15. Part-spores, larger-sized, more than 20 µm long 16
150. Part-spores, less than 20 µm long 17
16. Part-spores (12–)40–65 ×(3–)4–5 µmT. longisegmentatum
160. Part-spores 20–32 ×3.0–4.5 µmT. inusitaticapitatum
17. Part-spores, medium-sized, (13–)16(–21) ×2.5–3 µmT. rouxii
170. Part-spores, small-sized, 2.5–6 µm long 18
18. Perithecia elongate-ovoid, (560–)567–697(–750) ×(200–)206–248(–250) µm; part-spores 2–5 ×1.5–2 µmT. flavonigrum
180. Perithecia ovoid, 450–540 µm×230–260 µm; part-spores 3–6 (commonly 4.5) ×1.5–2 µmT. intermedium
19. Stromata clavate 20
190. Stromata capitate 21
20. Perithecia small, 245–495 µm long, deeply embedded; asci short, 195–270 µm long T. guangdongense
200. Perithecia 500–550 µm long, ostiola slightly projecting; asci 330–370 µm long T. japonicum
21. Perithecia large, more than 900 µm long 22
210. Perithecia medium-sized, 400–700 µm long 23
22. Perithecia ovoid, 900–1100 ×340–430 µm; part-spores cylindric or somewhat fusoid, 18–27 (commonly 24) ×2.5–3 µmT. capitatum
220. Perithecia ampullaceous, 900–930 ×220–250 µm; part-spores fusoid, 16–18 ×3µmT. minazukiense
23. Stipe slender, less than 1.0 mm thick 24
230. Stipe thick, columnar, 1.0–6.0 mm thick 25
24. Perithecia 500–600 ×250–350 µm; part-spores 2–5 ×1.5–2 µmT. fractum
240. Perithecia 400 ×250 µm; part-spores 6 ×1.5 µmT. virens
25. Asci 10–12 µm wide; part-spores medium-sized, 7.5–16 ×2.5–3 µmT. valvatistipitatum
250. Asci slender, 6–8 µm wide; part-spores small-sized, 3–8 ×2µmT. valliforme

Pathogens 2021,10, 1389 10 of 14
4. Materials and Methods
4.1. Collections and Morphology
Tolypocladium specimens, including their underground host Elaphomyces sp., were
collected in an evergreen broad-leaved forest in Lijiang Alpine Botanic Garden, Lijiang City,
Yunnan Province, China. The specimens were examined as described in Senanayake and
colleagues with the following modifications [
61
]. Colour codes were recorded following
those of Kornerup and Wanscher [
62
]. Specimens were deposited at the Herbarium of
Cryptogams Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
(HKAS, KUN).
4.2. DNA Extraction, PCR Amplification and Sequencing
The genomic DNA was extracted from the dried materials following the method de-
scribed by Dissanayake and colleagues [
63
]. Fertile tissues from the parasitic fungi and the
peridium of the host mushroom were used to extract DNA. Primer pairs ITS1F/ITS4 [
64
],
LR0R/LR5 [
65
,
66
], PNS1/NS8 [
64
], TEF1-
α
983F/TEF1-
α
2218R [
67
] and fRPB2-5F/fRPB2-
7R [
68
] were used for the amplification of the internal transcribed spacer region ITS1-5.8S-
ITS2 (ITS), the large subunit rDNA (LSU), the small subunit rDNA (SSU), the translation
elongation factor 1-
α
(TEF1-
α
) gene and RNA polymerase II second-largest subunit (RPB2),
respectively. PCR reaction was performed in a 25
µ
L reaction volume, comprising
12.5 µL
Taq PCR Master Mix (Abmgood, Richmond, BC, Canada), 1
µ
L forward primer, 1
µ
L
reverse primer, 2
µ
L DNA template and 8.5
µ
L ddH2O. For ITS, LSU, SSU and RPB2, PCR
reaction conditions were as follows: 5 min at 94
◦
C, followed by 35 cycles of 40 s at 94
◦
C,
40 s at 53
◦
C and 1 min at 72
◦
C, and a final extension of 10 min at 72
◦
C. PCR reaction
condition of TEF1-
α
was as follows: 5 min at 94
◦
C, followed by 35 cycles of 50 s at 94
◦
C,
40 s at 64
◦
C and 1 min at 72
◦
C, and a final extension of 10 min at 72
◦
C. The PCR products
were visualized using agarose gel electrophoresis after staining with dyes (TS-GelRed
Ver.2, Tsingke Biotechnology Co., Ltd., Beijing, China). Then, the products were sent for
sequencing at Sangon Biotech Co. Ltd., Shanghai, China.
4.3. Sequence Alignment and Phylogenetic Analyses
Phylogenetic trees were constructed using the sequencing data of T. inusitaticapi-
tatum and the allied reference sequences of closely related Ophiocordycipitaceae species
obtained from the GenBank (Table 5). Aschersonia confluens (BCC 7961) and A. para-
physata (BCC 1467) of Clavicipitaceae were used as outgroup taxa. All sequences were
assembled and aligned using MAFFT v 6.8 [
69
] and manually edited where necessary
in BioEdit version 7.0.9 [
70
]. Individual alignments were compiled for SSU, LSU, ITS,
TEF1-
α
and RPB2 genes. The optimal substitution model for each gene dataset was de-
termined using MrModeltest 2.3 [
71
] under the Akaike information criterion (AIC). The
results indicated that the GTR+I+G model was optimal for all the gene regions. Individ-
ual datasets were combined to assemble the combined dataset (gene order: SSU, LSU,
ITS, TEF1-
α
and RPB2). The resulted combined dataset was deposited in the TreeBASE
database (http://purl.org/phylo/treebase/phylows/study/TB2:S27887?x-access-code=
746eddc746009259527edd3d4c69526b&format=html, accessed on 10 March 2021).

Pathogens 2021,10, 1389 11 of 14
Table 5.
Voucher information and GenBank accession numbers for samples appearing in the Tolypocladium
phylogenetic tree
.
Taxon Strain/Specimen Voucher GenBank Accession Numbers
ITS 28S 18S TEF1-αRPB2
Aschersonia confluens BCC 7961 JN049841 DQ384947 DQ372100 DQ384976 DQ452465
A. paraphysata BCC 1467 DQ377987 DQ372090 DQ384967 DQ452463
Drechmeria gunnii OSC 76404 JN049822 AF339522 AF339572 AY489616 DQ522426
D. sinensis CBS 567.95 MH862540 AF339545 AF339594 DQ522343 DQ522443
D. zeospora ex CBS 335.80 MH861269 AF339540 AF339589 EF469062 EF469109
Ophiocordyceps gracilis EFCC 8572 JN049851 EF468811 EF468956 EF468751 EF468912
O. heteropoda EFCC 10125 JN049852 EF468812 EF468957 EF468752 EF468914
Paecilomyces lilacinus CBS 431.87 AY624188 EF468844 EF468791 EF468940
Pa. lilacinus ex CBS 284.36 AY624189 FR775484 EF468792 EF468941
Perennicordyceps cuboidea
NBRC 101740 JN943331 JN941417 JN941724 KF049684
Pe. cuboidea NBRC 100941 JN943329 JN941416 JN941725
Pe. paracuboidea NBRC 101742 JN943338 JN941431 JN941710 KF049685 KF049669
Pe. paracuboidea NBRC 100942 JN943337 JN941430 JN941711 AB972954 AB972958
Pe. prolifica TNS-F-18481 KF049659 KF049631 KF049612 KF049686
Pe. prolifica TNS-F-18547 KF049660 KF049632 KF049613 KF049687 KF049670
Polycephalomyces
aurantiacus MFLU 17-1393 MG136919 MG136913 MG136907 MG136877 MG136873
Po. aurantiacus MFLUCC 17 2113 MG136916 MG136910 MG136904 MG136875 MG136870
Po. marginaliradians MFLU 17-1582 MG136920 MG136914 MG136908 MG136878 MG271931
Po. marginaliradians MFLUCC 17-2276 MG1369 21 MG136915 MG136909 MG136879 MG271930
Po. nipponicus NBRC 101406 JN943301 JN941388 JN941753
Po. nipponicus BCC 1682 KF049664 KF049638 KF049620 KF049694 MF416463
Po. yunnanensis YHCPY1005 KF977848 KF977848 KF977848 KF977850 KF977854
Po. yunnanensis YHHPY1006 KF977849 KF977849 KF977849 KF977851 KF977855
Tolypocladium
amazonense VPB179 KF747267 KF747329
T. amazonense ex MS308 KF747134 KF747314 KF747099
T. capitatum NBRC 106325 JN941402 JN941739 AB968598 AB968559
T. capitatum NBRC 100997 JN941401 JN941740 AB968597 AB968558
T. cylindrosporum ARSEF 2920 MG228381 MG228390 MG228387
T. cylindrosporum YFCC 1805001 MK984581 MK984577 MK984565 MK984569 MK984573
T. endophyticum MX535 KF747260 KF747153 KF747322 KF747117
T. flavonigrum ex BCC 66576 MN338090 MN337287 MN338495
T. flavonigrum BCC 66578 MN338091 MN337288 MN338496
T. flavonigrum BCC66580 MN337289 MN338497
T. fractum OSC 110990 DQ518759 DQ522545 DQ522328 DQ522425
T. fumosum WA18945 KU925171 KU985053
T. geodes CBS 126054 MH864065 MH875520
T. inflatum OSC 71235 JN049844 EF469077 EF469124 EF469061 EF469108
T. inflatum CBS 127302 MH864514 MH875949
T. inusitaticapitatum HKAS 112152 MW537735 MW537718 MW537733 MW507527 MW507529
T. inusitaticapitatum HKAS 112153 MW537736 MW537719 MW537734 MW507528 MW507530
T. jezoense txid94205 AB027365 AB027365 AB027319
T. longisegmentatum OSC 110992 EF468816 EF468919
T. nubicola CBS 568.84 MH861780 MH873478
T. ophioglossoides CBS 100239 KU382155 KJ878874 KJ878910 KJ878958
T. ophioglossoides NBRC 8992 JN943316 JN941405 JN941736 AB968601 AB968562
T. ovalisporum CBS 700.92 AB457006
T. paradoxum NBRC 106958 JN943324 JN941411 JN941730 AB968600 AB968561
T. paradoxum NBRC 100945 JN943323 JN941410 JN941731 AB968599 AB968560
T. pustulatum MRL GB6597 AF389189 AF389190
T. tropicale MX338 KF747259 KF747149 KF747318 KF747113
T. tropicale ex IQ214 KF747254 KF747125 KF747090
T. tundrense CBS 569.84 MH861781 MH873479
T. valliforme DAOM 196368 AY245640 AY245648
New sequencing data are displayed in bold. Specimens of the current study are given in red. Type specimens are in bold; superscript ‘ex’
indicates ex-type.
Maximum likelihood (ML) analysis was performed using IQ-Tree (http://iqtree.cibiv.
univie.ac.at/, accessed on 20 May 2021) [
72
,
73
]. The substitution model options for each
gene were auto-evaluated according to the provided partition file. Clade support for the
ML analysis was assessed using an SH-aLRT test with 1000 replicates [
74
] and the ultrafast
bootstrap (UFB) [
75
]. In the ML analyses, nodes with support values of SH-aLRT
≥
80 and
UFB
≥
95 were considered well-supported, those with either SH-aLRT < 80 or UFB < 95
were considered weakly supported, and nodes with SH-aLRT < 80 and UFB < 95 were
considered unsupported.
Bayesian Inference (BI) analysis was carried out in MrBayes v3.2.6 [
76
]. Gaps were
treated as missing data. Four simultaneous Markov Chain Monte Carlo (MCMC) chains
were run for 10,000,000 generations and were sampled at every 100th generation until
the standard deviation of the split frequencies fell below 0.01 and ESS values > 200. Sub-
sequently, phylogenetic trees were summarized and posterior probabilities (PP) were

Pathogens 2021,10, 1389 12 of 14
calculated using MCMC by discarding the first 25% generations as the burn-in phase [
77
].
Phylogenetic trees were viewed in FigTree v.1.4.4. Nodes with BI posterior probability
(BIPP) > 0.90 were considered to be well supported.
Author Contributions:
This study was initiated by F.-M.Y. and K.D.H. Samples were collected by
J.-W.L. Morphological observation and description were done by F.-M.Y., K.D.H., K.W.T.C., D.-P.W.,
S.-M.T., J.-W.L. and L.L., and phylogeny analyses were done by F.-M.Y., K.W.T.C. and Q.Z. The
manuscript was mainly drafted by F.-M.Y. with contributions from all other authors. All authors
have read and agreed to the published version of the manuscript.
Funding:
This research is supported by the National Natural Science Foundation of China, grant
“Characterization of roots and their associated rhizosphere microbes in agroforestry systems: ecologi-
cal restoration in high-phosphorus environment” (Grant No. 31861143002), “Impact of climate change
on fungal diversity and biogeography in the Greater Mekong Subregion” (Grant No. RDG6130001),
the Open Research Project “Cross-Cooperative Team” of the Germplasm Bank of Wild Species,
Kunming Institute of Botany, Chinese Academy of Sciences (Grant No. 292019312511043), Guizhou
Science and Technology Planning Project (Guizhou Science and Technology Cooperation Support
[2021] General 200) and the Joint Agricultural Program of Yunnan Province (No.2018FG001 032).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The obtained gene sequences were deposited in the NCBI GenBank
database. The accession numbers of the obtained sequences are contained within the article and
in Table 5.
Acknowledgments:
The authors would like to express their sincere thanks to Ling-Sheng Zha (School
of Life Sciences, Huaibei Normal University, Huaibei 235000, People’s Republic of China) for his help
in finding literature and comments on this work; to Shaun Pennycook (Manaaki Whenua—Landcare
Research, Private Bag 92170, Auckland 1072, New Zealand) for nomenclatural review; to Jennifer
Luangsa-ard (National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand
Science Park, Phahonyothin Rd., Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand) for
providing detailed information on Tolypocladium flavonigrum.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Sun, J.-Z.; Liu, X.-Z.; McKenzie, E.H.; Jeewon, R.; Liu, J.-K.; Zhang, X.-L.; Zhao, Q.; Hyde, K.D. Fungicolous fungi: Terminology,
diversity, distribution, evolution, and species checklist. Fungal Divers. 2019,95, 337–430. [CrossRef]
2.
Wijayawardene, N.N.; Hyde, K.D.; Al-Ani, L.K.T.; Tedersoo, L.; Haelewaters, D.; Rajeshkumar, K.C.; Zhao, R.L.; Aptroot, A.;
Leontyev, D.V.; Saxena, R.K.; et al. Outline of Fungi and fungus-like taxa. Mycosphere 2020,11, 1060–1456. [CrossRef]
3.
Hyde, K.D.; Norphanphoun, C.; Maharachchikumbura, S.S.N.; Bhat, D.J.; Jones, E.B.G.; Bundhun, D.; Chen, Y.J.; Bao, D.F.;
Boonmee, S.; Calabon, M.S.; et al. Refined families of Sordariomycetes. Mycosphere 2020,11, 305–1059. [CrossRef]
4. Gams, W. Tolypocladium, eine Hyphomycetengattung mit geschwollenen Phialiden. Persoonia 1971,6, 185–191.
5.
Hodge, K.T.; Krasnoff, S.B.; Humber, R.A. Tolypocladium inflatum is the anamorph of Cordyceps subsessilis.Mycologia
1996
,88,
715–719. [CrossRef]
6.
Sung, G.H.; Hywel-Jones, N.L.; Sung, J.M.; Luangsa-ard, J.J.; Shrestha, B.; Spatafora, J.W. Phylogenetic classification of Cordyceps
and the clavicipitaceous fungi. Stud. Mycol. 2007,57, 5–59. [CrossRef] [PubMed]
7.
Gams, W. Chaunopycnis alba, gen. et sp. nov., a soil fungus intermediate between Moniliales and Sphaeropsidales. Persoonia
1980
,
11, 75–79.
8.
Quandt, C.A.; Kepler, R.M.; Gams, W.; Araújo, J.P.M.; Ban, S.; Evans, H.C.; Hughes, D.; Humber, R.; Hywel-Jones, N.; Li, Z.; et al.
Phylogenetic-based nomenclatural proposals for Ophiocordycipitaceae (Hypocreales) with new combinations in Tolypocladium.
IMA Fungus 2014,5, 121–134. [CrossRef]
9. Mains, E.B. Species of Cordyceps parasitic on Elaphomyces.Bull. Torrey Bot. Club 1957,84, 243–251. [CrossRef]
10.
Kobayasi, Y.; Shimizu, D. Monographic studies of Cordyceps 1, group parasitic on Elaphomyces.Bull. Natl. Sci. Mus. Tokyo
1960
,
5, 69–85.
11.
Nikoh, N.; Fukatsu, T. Interkingdom host jumping underground: Phylogenetic analysis of entomoparasitic fungi of the genus
Cordyceps.Mol. Biol. Evol. 2000,17, 629–638. [CrossRef]
12.
Gazis, R.; Skaltsas, D.; Chaverri, P. Novel endophytic lineages of Tolypocladium provide new insights into the ecology and
evolution of Cordyceps-like fungi. Mycologia 2014,106, 1090–1105. [CrossRef] [PubMed]

Pathogens 2021,10, 1389 13 of 14
13.
Sung, G.H.; Sung, J.M.; Hywel-Jones, N.L.; Spatafora, J.W. A multi-gene phylogeny of Clavicipitaceae (Ascomycota, Fungi):
Identification of localized incongruence using a combinational bootstrap approach. Mol. Phylogenet. Evol.
2007
,44, 1204–1223.
[CrossRef] [PubMed]
14.
Kepler, R.; Ban, S.; Nakagiri, A.; Bischoff, J.; Hywel-Jones, N.; Owensby, C.A.; Spatafora, J.W. The phylogenetic placement of
hypocrealean insect pathogens in the genus Polycephalomyces: An application of One Fungus One Name. Fungal Biol.
2013
,117,
611–622. [CrossRef]
15.
Quandt, C.A.; Patterson, W.; Spatafora, J.W. Harnessing the power of phylogenomics to disentangle the directionality and
signatures of interkingdom host jumping in the parasitic fungal genus Tolypocladium.Mycologia 2018,110, 104–117. [CrossRef]
16.
Quandt, C.A.; Di, Y.; Elser, J.; Jaiswal, P.; Spatafora, J.W. Differential expression of genes involved in host recognition, attachment,
and degradation in the mycoparasite Tolypocladium ophioglossoides.G3 Genes Genomes Genet. 2016,6, 731–741. [CrossRef]
17.
Wijayawardene, N.N.; Hyde, K.D.; Rajeshkumar, K.C.; Hawksworth, D.L.; Madrid, H.; Kirk, P.M.; Braun, U.; Singh, R.V.;
Crous, P.W.; Kukwa, M.; et al. Notes for genera: Ascomycota. Fungal Divers. 2017,86, 1–594. [CrossRef]
18.
Wang, J.-C.; Zhang, Z.-Z.; Li, C.-L.; Wang, Y. Research Progress of Tolypocladium in Ophiocordycipitaceae. J. Fungal Res.
2019
,
1–10. [CrossRef]
19. Maas Geesteranus, R.A. On ‘Cordyceps capitata’. Persoonia 1963,2, 477–482.
20.
Ginns, J. Typification of Cordyceps canadensis and C. capitata, and a new species, C. longisegmentis.Mycologia
1988
,80,
217–222. [CrossRef]
21. Zang, M.; Kinjo, N. Notes on the alpine Cordyceps of China and nearby nations. Mycotaxon 1998,66, 215–229.
22.
Zhang, D.-Z.; Zhou, W.-N.; Wu, S.-H.; Wang, Y.-Z. Macrofungi in Fushan; Council of Agriculture, Executive Office:
Taipei, Taiwan, 2000.
23. Bissett, J. Notes on Tolypocladium and related genera. Can. J. Bot. 1983,61, 1311–1329. [CrossRef]
24.
Samson, R.A.; Soares, G.G. Entomopathogenic species of the hyphomycete genus Tolypocladium.J. Invertebr. Pathol.
1984
,43,
133–139. [CrossRef]
25.
Wright, D.A.; Cummings, N.J.; Haack, N.A.; Jackson, T.A. Tolypocladium cylindrosporum, a novel pathogen for sheep blowflies.
New Zeal. J. Agric. Res. 2009,52, 315–321. [CrossRef]
26.
Xu, W.-S.; LYU, G.-Z.; Jiang, H.; Zhao, Z.-H.; Sun, X.-D.; LYU, S.-Y. Three species of Tolypocladium isolated from forest soil of
Changbai mountain. J. Fungal Res. 2012,10, 143–146.
27.
Montalva, C.; Silva, J.J.; Rocha, L.F.N.; Luz, C.; Humber, R.A. Characterization of Tolypocladium cylindrosporum (Hypocreales,
Ophiocordycipitaceae) isolates from Brazil and their efficacy against Aedes aegypti (Diptera, Culicidae). J. Appl. Microbiol.
2019
,
126, 266–276. [CrossRef] [PubMed]
28. Liang, Z.-Q.; Liu, A.-Y.; Liu, M.-H. Two new records of mycogenous Cordyceps in China. Mycosystema 2003,22, 159–160.
29.
Li, C.; Hywel-jones, N.; Cao, Y.; Nam, S.; Li, Z. Tolypocladium dujiaolongae sp. nov. and its allies. Mycotaxon
2018
,133,
229–241. [CrossRef]
30.
Crous, P.W.; Cowan, D.A.; Yilmaz, N.; Larsson, E.; Angelini, C.; Brandrud, T.E.; Dearnaley, J.D.W.; Dima, B.; Dovana, F.;
Fechner, N.; et al. Fungal Planet description sheets: 1112–1181. Persoonia 2020,45, 251–409. [CrossRef]
31.
Crous, P.W.; Wingfield, M.J.; Burgess, T.I.; Hardy, G.E.S.J.; Barber, P.A.; Alvarado, P.; Barnes, C.W.; Buchanan, P.K.; Heykoop, M.;
Moreno, G.; et al. Fungal Planet description sheets: 558–624. Persoonia 2017,38, 240–384. [CrossRef] [PubMed]
32. Lin, Q.-Y.; Li, T.-H.; Song, B. Cordyceps guangdongensis sp. nov. from China. Mycotaxon 2008,103, 371–376.
33. Liang, Z.-Q. Flora Fungorum Sinicorum. Cordyceps; Science Press: Beijing, China, 2007; Volume 32.
34.
Kobayasi, Y.; Shimizu, D. Monographic studies of Cordyceps 2, group parasitic on cicadae. Bull. Natl. Sci. Mus. Tokyo
1963
,
6, 286–314.
35.
Suo, F.-Y.; Huang, L.-D.; Yu, H. Identification and antibacterial effect research of a Tolypocladium strain isolated from sclerotium of
Ophiocordyceps gracilis in Xinjiang. China J. Chin. Mater. Med. 2014,39, 965–971.
36. Imai, S. On a new species of Cordyceps parasitic on Elaphomyces in Japan. Proc. Imp. Acad. 1934,10, 677–679. [CrossRef]
37. Ke, Y.-H.; Ju, Y.-M. Two rare ophiocordycipitaceous fungi newly recorded in Taiwan. Bot. Stud. 2015,56. [CrossRef]
38.
Barron, G.L. Structure and biology of a new Tolypocladium attacking bdelloid rotifers. Can. J. Bot.
1983
,61, 2566–2569. [CrossRef]
39. Zeng, X.-L.; Yang, W.-S. Cordyceps canadensis a new record in China. Edible Fungi China 1990,9, 27.
40. Kobayasi, Y.; Shimizu, D. Cordyceps species from Japan 5. Bull. Natl. Sci. Mus. Tokyo Ser. B Bot. 1982,8, 111–123.
41. Han, Y.-F.; Liang, Z.-Q.; Chu, H.-L. Tolypocladium nubicola, a new record of Tolypocladium in China. J. Fungal Res. 2004,2, 50–52.
42.
Sung, J.M.; Choi, Y.S.; Kim, Y.O.; Kim, S.H.; Sung, G.H. Cordyceps species collected by Korean entomopathogenic fungal
collection. In Proceedings of the Third Korean-China Joint Symposium for Mycology (The Korean Society of Mycology and
Chinese Academy of Sciences), Seoul, Korea, December 1997; The Korean Society of Mycology: Seoul, Korea, 1997; pp. 49–60.
43. Lee, T.S.; Yoon, K.H. The Index of Korea-Japan Mushroom Names in Korea; Personal Printing: Seoul, Korea, 2002.
44.
Song, B.; Lin, Q.-Y.; Li, T.-H.; Shen, Y.-H.; Li, J.-J.; Luo, D.-X. Known species of Cordyceps from China and their distribution. J.
Fungal Res. 2006,4, 10–26.
45. Möller, C.; Gams, W. Two new hyphomycetes isolated from Antarctic lichens. Mycotaxon 1993,48, 441–450.
46.
Zha, L.-S.; Xiao, Y.-P.; Jeewon, R.; Zou, X.; Wang, X.; Boonmee, S.; Eungwanichayapant, P.D.; McKenzie, E.H.C.; Hyde, K.D.;
Wen, T.-C. Notes on the medicinal mushroom chanhua (Cordyceps cicadae (Miq.) Massee). Chiang Mai J. Sci. 2019,46, 1023–1035.

Pathogens 2021,10, 1389 14 of 14
47.
Chen, Z.-A.; Li, Z.-Z.; Chen, Y.-P. Jin-Chanhua (A Precious Cordyceps: Isaria cicadae Miq.); Publishing House of Ancient Chinese
Medical Books: Beijing, China, 2014.
48.
Bills, G.F.; Polishook, J.D.; Goetz, M.A.; Sullivan, R.F.; White, J.F. Chaunopycnis pustulata sp. nov., a new clavicipitalean anamorph
producing metabolites that modulate potassium ion channels. Mycol. Prog. 2002,1, 3–17. [CrossRef]
49. Teng, S.C. Additional fungi from China IV. Sinensia 1936,7, 752–823.
50. Teng, S.C. Fungi of China; Korf, R.P., Ed.; Mycotaxon Ltd.: Ithaca, NY, USA, 1996; pp. 118–125.
51. Candoussau, F. Un Cordyceps nouveau des Pyrenees Francaises: Cordyceps Rouxii sp. nov. Mycotaxon 1976,IV, 540–544.
52. Li, C.-L. Study of a new species of Tolypocladium.J. Nanjing Univ. (Nat. Sci. Ed.) 1986,22, 401–407, 592.
53. Zang, M. Two New Tropical Mycotaxon from Yunnan, China. Acta Bot. Yunnanica 2001,23, 295–297.
54.
Weiser, J.; Matha, V.; Jegorov, A. Tolypocladium terricola sp. n., a new mosquito-killing species of the genus Tolypocladium Gams
(Hyphomycetes). Folia Parasitol. 1991,38, 363–369.
55. Barron, G.L. Two new fungal parasites of bdelloid rotifers. Can. J. Bot. 1981,59, 1449–1455. [CrossRef]
56. Kobayasi, Y. Miscellaneous notes of fungi (3). J. Jpn. Bot. 1983,58, 221–224.
57.
Sung, G.H.; Poinar, G.O.; Spatafora, J.W. The oldest fossil evidence of animal parasitism by fungi supports a Cretaceous
diversification of fungal-arthropod symbioses. Mol. Phylogenet. Evol. 2008,49, 495–502. [CrossRef]
58.
Spatafora, J.W.; Sung, G.H.; Sung, J.M.; Hywel-Jones, N.L.; White, J.F. Phylogenetic evidence for an animal pathogen origin of
ergot and the grass endophytes. Mol. Ecol. 2007,16, 1701–1711. [CrossRef]
59.
Bushley, K.E.; Raja, R.; Jaiswal, P.; Cumbie, J.S.; Nonogaki, M.; Boyd, A.E.; Owensby, C.A.; Knaus, B.J.; Elser, J.; Miller, D.; et al.
The Genome of Tolypocladium inflatum: Evolution, organization, and expression of the Cyclosporin biosynthetic gene cluster. PLoS
Genet. 2013,9, e1003496. [CrossRef] [PubMed]
60.
Quandt, C.A.; Bushley, K.E.; Spatafora, J.W. The genome of the truffle-parasite Tolypocladium ophioglossoides and the evolution of
antifungal peptaibiotics. BMC Genom. 2015,16, 1–14. [CrossRef] [PubMed]
61.
Senanayake, I.C.; Rathnayaka, A.R.; Marasinghe, D.S.; Calabon, M.S.; Gentekaki, E.; Lee, H.B.; Hurdeal, V.G.; Pem, D.;
Dissanayake, L.S.;
Wijesinghe, S.N.; et al. Morphological approaches in studying fungi: Collection, examination, isolation,
sporulation and preservation. Mycosphere 2020,11, 2678–2754. [CrossRef]
62. Kornerup, A.; Wanscher, J.H. Methuen Handbook of Colour, 3rd ed.; Eyre Methuen: London, UK, 1978.
63.
Dissanayake, A.J.; Bhunjun, C.S.; Maharachchikumbura, S.S.N.; Liu, J.-K. Applied aspects of methods to infer phylogenetic
relationships amongst fungi. Mycosphere 2020,11, 2652–2676. [CrossRef]
64.
White, T.J.; Bruns, T.; Lee, S.J.W.T.; Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA Genes for phylogenetics.
PCR Protoc. A Guide Methods Appl. 1990,18, 315–322. [CrossRef]
65.
Rehner, S.A.; Samuels, G.J. Taxonomy and phylogeny of Gliocladium analysed from nuclear large subunit ribosomal DNA
sequences. Mycol. Res. 1994,98, 625–634. [CrossRef]
66.
Vilgalys, R.; Hester, M. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several
Cryptococcus species. J. Bacteriol. 1990,172, 4238–4246. [CrossRef] [PubMed]
67.
Rehner, S.A.; Buckley, E. A Beauveria phylogeny inferred from nuclear ITS and EF1-
α
sequences: Evidence for cryptic diversification
and links to Cordyceps teleomorphs. Mycologia 2005,97, 84–98. [CrossRef] [PubMed]
68.
Liu, Y.J.; Whelen, S.; Hall, B.D. Phylogenetic relationships among ascomycetes: Evidence from an RNA polymerse II subunit. Mol.
Biol. Evol. 1999,16, 1799–1808. [CrossRef]
69.
Katoh, K.; Kuma, K.I.; Toh, H.; Miyata, T. MAFFT version 5: Improvement in accuracy of multiple sequence alignment. Nucleic
Acids Res. 2005,33, 511–518. [CrossRef]
70.
Hall, T.A. BIOEDIT: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic
Acids Symp. Ser. 1999,41, 95–98.
71. Nylander, J.A. MrModeltest 2. Program distributed by the author. Evol. Biol. Cent. Upps. Univ. 2004, 2–4.
72.
Nguyen, L.T.; Schmidt, H.A.; Von Haeseler, A.; Minh, B.Q. IQ-TREE: A fast and effective stochastic algorithm for estimating
maximum-likelihood phylogenies. Mol. Biol. Evol. 2015,32, 268–274. [CrossRef] [PubMed]
73.
Chernomor, O.; Von Haeseler, A.; Minh, B.Q. Terrace aware data structure for phylogenomic inference from Supermatrices. Syst.
Biol. 2016,65, 997–1008. [CrossRef]
74.
Guindon, S.; Dufayard, J.F.; Lefort, V.; Anisimova, M.; Hordijk, W.; Gascuel, O. New algorithms and methods to estimate
maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Syst. Biol.
2010
,59, 307–321. [CrossRef] [PubMed]
75.
Hoang, D.T.; Chernomor, O.; Von Haeseler, A.; Minh, B.Q.; Vinh, L.S. UFBoot2: Improving the ultrafast bootstrap approximation.
Mol. Biol. Evol. 2018,35, 518–522. [CrossRef]
76.
Ronquist, F.; Teslenko, M.; Van Der Mark, P.; Ayres, D.L.; Darling, A.; Höhna, S.; Larget, B.; Liu, L.; Suchard, M.A.;
Huelsenbeck, J.P.
Mrbayes 3.2: Efficient bayesian phylogenetic inference and model choice across a large model space. Syst. Biol.
2012
,61,
539–542. [CrossRef]
77. Huelsenbeck, J.P.; Ronquist, F. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 2001,17, 754–755. [CrossRef]