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.

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 ecosystems 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 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 ectomycorrhizal 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 [4,6,7,8].

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 [6,8,11,12,13,14,15]. 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 recognition, attachment, and then infection and degradation, depending on the gene content, expression, or regulation [16]. Tolypocladium is recognized as an ideal candidate for investigating the mechanisms associated with host-jumping [15,16]. Quandt and colleagues researched the set of genes that are differentially regulated in Tolypocladium species during 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 media 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 distribution [2,17]. Some of them produce various secondary metabolites, such as cyclosporin, efrapeptins, ophiocordin and ophiosetin [18]. They have been widely used in biopharmaceuticals 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.

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.) [9,10,19,20,21,22]
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. [4,23,24,25,26,27]
* 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 [9,10,42,43,44]
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).

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).

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.

Table 2.

Intraspecific base-pair differences in LSU genes among Tolypocladium species.

Locus

522

532

855

Ratio

Species

T. album

CBS 393.89 #

C

C

C

0.35% (3/870 bp)

GB5502

T

T

-

Locus

20

21

23

24

25

27

Ratio

Species

T. inflatum

OSC 71235 #

A

G

A

A

C

A

0.76% (6/794 bp)

CBS 127302

G

A

-

-

-

C

Locus

48

434

Ratio

Species

T. ophioglossoides

CBS 100239 #

C

C

0.25% (2/816 bp)

NBRC 106330

T

T

Locus

164

382

405

433

442

479

496

524

Ratio

Species

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

Locus

8

37

44

51

81

96

110

124

204

210

402

Ratio

Species

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 distribution), 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.

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 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 (including the novel species described in this study) distributed worldwide [2,3,17]. Seventeen 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.

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) = 2 cm; (e) = 2 mm; (f) = 500 μm; (g) = 50 μm; (h) = 100 μm; (i–n) = 250 μm; (o–q) = 20 μm.

Index Fungorum: IF558123; Facesoffungi number: FoF 10407.

Typification: China, Yunnan Province, Lijiang City, Lijiang Alpine Botanic Garden, E100°10′58.07, N26°59′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 directly 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 pseudoparenchymatous 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 μm in high) or invisible, lined with periphyses. Asci is 410–510 μm × 10–15 μm (x = 461 μm × 13 μm, n = 20), hyaline, and long cylindrical, with a conspicuously thickened cap (measuring 6.5–7.5 μm × 6.0–7.0 μm). Ascospores are approximately as long as asci, and extremely easy to break into part-spores. Part-spores 20–32 μm × 3.0–4.5 μm (x = 25 μm × 3.6 μm, n = 20), hyaline, cylindrical with rounded ends. Asexual morph: Unknown.

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).

Notes: Based on the multi-gene phylogeny results, our specimens are closely related to Tolypocladium flavonigrum, known only from Thailand. Both species have stromata directly emerging from the surface of Elaphomyces sp., and capitate fertile heads with the perithecia entirely immersed in a well-differentiated valliforme-like structure [30]. However, 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 μm × 10–15 μm, 20–32 μm × 3.0–4.5 μm) 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 μm wide) [10]. Tolypocladium fractum differs from T. inusitaticapitatum 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 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 [8,9,10]. 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
1′. Host hypogeous Elaphomyces spp.	7
2. Host beetle or moth larvae	3
2′. Host cicada nymphs	4
3. Fertile part capitate, with stellate appearance; perithecia ovoid to pear-shaped, 740–760 × 444–558 μm	T. fumosum
3′. Fertile part, strap-shaped pseudostalk; perithecia superficial, narrow flask-shaped, 1000–1500 × 330–440 μm	T. inflatum
4. Stromata arising from underground mycelial membrane or strand; part-spores 3–5 × 1.5–2 μm	T. paradoxum
4′. 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
5′. Fertile part oblong or clavate	6
6. Perithecia superficial or apparently half-immersed, pyriform, 520–550 × 260–280 μm; part-spores 2.5–3 × 2 μm	T. inegoense
6′. Perithecia wholly immersed, ampullaceous, (233–)520–740(–780) × (250–)300–330 (–360) μm; part-spores 3–5(–7.0) × 2–3 μm	T. dujiaolongae
7. Stroma attached to host by rhizomorphs	8
7′. Stroma arising directly from the host	12
8. Part-spores articulate, moniliform, 3–3.5 × 2–2.5 μm	T. szemaoense
8′. Part-spores with truncate or rounded ends	9
9. Stroma capitate	10
9′. Stroma solitary or rarely caespitose	11
10. Perithecia small, 480–540 × 225–255 μm; part-spores large-sized, 18–28 × 3–5 μm	T. delicatistipitatum
10′. Perithecia 770–800 × 350–430 μm; part-spores medium-sized, 8–11 × 1.5–2 μm	T. miomoteanum
11. Perithecia oblong with long neck, 700–720 × 200–250 μm; part-spores long, 20–30(50) × 3–4.5 μm	T. jezoense
11′. Perithecia ovoid, 550–600 × 200–300 μm; part-spores small short rod-shaped, 2.5–5 × 1.5–2 μm	T. ophioglossoides
12. Perithecia superficial, ascospores nonfractured	T. ramosum
12′. Perithecia entirely embedded or ostiole slightly projecting	13
13. Fertile part, cortex composed of pseudoparenchymatous peridial layer, and with an ectal layer	14
13′. 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 μm	T. tenuisporum
14′. Stromata capitate	15
15. Part-spores, larger-sized, more than 20 μm long	16
15′. Part-spores, less than 20 μm long	17
16. Part-spores (12–)40–65 × (3–)4–5 μm	T. longisegmentatum
16′. Part-spores 20–32 × 3.0–4.5 μm	T. inusitaticapitatum
17. Part-spores, medium-sized, (13–)16(–21) × 2.5–3 μm	T. rouxii
17′. 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 μm	T. flavonigrum
18′. Perithecia ovoid, 450–540 μm × 230–260 μm; part-spores 3–6 (commonly 4.5) × 1.5–2 μm	T. intermedium
19. Stromata clavate	20
19′. Stromata capitate	21
20. Perithecia small, 245–495 μm long, deeply embedded; asci short, 195–270 μm long	T. guangdongense
20′. Perithecia 500–550 μm long, ostiola slightly projecting; asci 330–370 μm long	T. japonicum
21. Perithecia large, more than 900 μm long	22
21′. 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 μm	T. capitatum
22′. Perithecia ampullaceous, 900–930 × 220–250 μm; part-spores fusoid, 16–18 × 3 μm	T. minazukiense
23. Stipe slender, less than 1.0 mm thick	24
23′. Stipe thick, columnar, 1.0–6.0 mm thick	25
24. Perithecia 500–600 × 250–350 μm; part-spores 2–5 × 1.5–2 μm	T. fractum
24′. Perithecia 400 × 250 μm; part-spores 6 × 1.5 μm	T. virens
25. Asci 10–12 μm wide; part-spores medium-sized, 7.5–16 × 2.5–3 μm	T. valvatistipitatum
25′. Asci slender, 6–8 μm wide; part-spores small-sized, 3–8 × 2 μm	T. valliforme

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 described 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 μL reaction volume, comprising 12.5 μL Taq PCR Master Mix (Abmgood, Richmond, BC, Canada), 1 μL forward primer, 1 μL reverse primer, 2 μL DNA template and 8.5 μL 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. inusitaticapitatum and the allied reference sequences of closely related Ophiocordycipitaceae species obtained from the GenBank (Table 5). Aschersonia confluens (BCC 7961) and A. paraphysata (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 determined 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. Individual 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).

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-α	RPB2
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. Subsequently, phylogenetic trees were summarized and posterior probabilities (PP) were 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: ecological 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.

Conflicts of Interest

The authors declare no conflict of interest.