Multiple genotypes of Ophiocordyceps sinensis in ...

Multiple genotypes of Ophiocordyceps sinensis in natural Cordyceps sinensis insect-fungi complex ZHU Jia-Shi 1,2* 1

LI Yu-Ling 3,4

YAO Yi-Sang 1

WU Jian-Yong 2,5

XIE Wei-Dong 1

Tsinghua University Graduate School at Shenzhen, Guangdong 518055, China; 2 The Hong Kong Polytechnic University, Hong Kong; 3

Qinghai University, Xining, Qinghai 810016, China; Veterinary Sciences, Qinghai 810016, China;

5

4

Institute of Grassland Research, Qinghai Academy of Animal Husbandry and

State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Shenzhen

Guangdong 518057, China

Abstract: Detections of 17 genotypes of Ophiocordyceps sinensis have been reported in studies of natural Cordyceps sinensis that consists of multiple intrinsic fungi and a dead larva from the Family Hepialidae. However, the multiple genotypes of O. sinensis fungi, as well as the natural insect-fungi complex, indiscriminately share the same Latin name. This paper reviews the scientific findings of multiple genotypes of O. sinensis in natural C. sinensis and related hypotheses, and also lists suggestions for renaming. This paper encourages the cooperation of taxonomists across disciplines to taxonomically characterize the multiple mutant genotype fungi and to resolve the taxonomy-nomenclature problem to end the centuries-old academic confusion and debate on the indiscriminate use of the Latin name O. sinensis for O. sinensis fungi and the natural insect-fungi complex. Key words: Natural Cordyceps sinensis insect-fungi complex; mutant genotypes of Ophiocordyceps sinensis fungi; heterokaryon; genetic heterogeneity

Leung et al. 2006, Stensrud et al. 2007, Yang et al.

1 INTRODUCTION Natural Cordyceps sinensis is defined as an insect-fungi

complex

according

to

Chinese

Pharmacopeia and scientific papers from which diverse anamorphic fungi (>90 species spanning more than 37 genera) have been identified (Jiang & Yao 2002, 2005, He et al. 2010, 2011, Zhang et al. 2010, Xia et al. 2015). Mycology and molecular mycology

studies

have

identified

multiple

Ophiocordyceps sinensis fungi (Chen et al. 2004,

2008, Xiao et al. 2009, Zhu et al. 2010, 2012, 2016, Barseghyan et al. 2011, Yao et al. 2011, Li et al. 2013, 2016d, Mao et al. 2013, Wei et al. 2016). The International presented

its

Fungus=One

Mycological Amsterdam Name”

Nomenclature

with for

the

fungal

in

“One

2011

and

nomenclature in

International

algae,

(IMA)

Declaration,

(1F=1N),

established rules for combination

Association

fungi,

Code and

of

plants

(Hawksworth et al. 2011, Taylor 2011, McNeill et al.

Supported by Ministry of Science and Technology of China (No. 2013BAD16B012: Studies on screening of high-quality lines of insect (the family Hepialidae) for Cordyceps sinensis and construction of a demonstration base). * Corresponding author. E-mail: [email protected] Received: 11-07-2017, accepted: xx-xx-2017

2

Submitted to CORDYCEPS FORUM 2017, Shenyang, Liaoning, China (August 5-7, 2017)

2012). 1F=1N states that one name can be used for

et al. (2011) reported the 2 anamorphs (H. sinensis

both the anamorph and teleomorph (nomenclature) of

and

only a single fungal species (taxonomic prerequisite)

Additional psychrophilic fungal species have also

to avoid any abuse of 1F=1N in a situation in which

been identified from natural C. sinensis (Jiang & Yao

the same name is applied to multiple fungi. O.

2005, He et al. 2010, 2011). Zhang et al. (2010) and

sinensis represents a special case in which the Latin

Xia et al. (2015) identified more than 90 fungal

name refers to multiple teleomorphic and anamorphic

species or operational taxonomic units (OUTs)

fungi (multiple fungal species and multiple genotypes

spanning at least 37 fungal genera and reported

of O. sinensis), as well as to the natural insect-fungi

different dominant fungal species, but H. sinensis was

complex DōngChóngXìaCăo 冬虫夏草 (natural C.

not detected from the stroma or caterpillar body of

sinensis hereinafter) (Zhang et al. 2012, Ren et al.

natural C. sinensis.

2013, Zhu & Wu 2015, Yao & Zhu 2016, Li et al.

2C. Hypothesis III: O. sinensis is a collective term

2016c, Zhu & Li 2017).

for multiple mutant genotypes of fungi (Chen et al.

2 DOES O. SINENSIS REFER TO A FUNGUS OR TO A GROUP OF FUNGI? The first fundamental question in the academic debate is whether O. sinensis is a fungus or a group of fungi and thus if 1F=1N is applicable to O. sinensis research. There are 3 controversial hypotheses reported in the literature (Zhu & Wu 2015, Li et al. 2016c, Yao & Zhu 2016, Zhu & Li 2017). 2A. Hypothesis I: O. sinensis is a single fungus (Wei et al. 2006, Guo et al. 2010) Liu et al. (1989) reported the identification of Hirsutella sinensis from natural C. sinensis and set the H. sinensis strain 8706-(2)870525(III) (dry type strain HMAS 55469) as the type material for the fungus. Prof. Y-L Guo (the second naming author) and coauthors stated in (Guo et al. 2010) (translated) “DōngChóngXìaCăo is a fungal species … The

Tolypocladium

sinensis)

of

O.

sinensis.

2004, Stensrud et al. 2007, Yang et al. 2008, Xiao et al. 2009, Zhang et al. 2009, 2013a, Zhu et al. 2010, 2012, 2016, Gao et al. 2011, 2012, Yao et al. 2011, Li et al. 2013, 2016b, d, Mao et al. 2013, Wei et al. 2016) Stensrud et al. (2007) reported 3 phylogenetic clades of O. sinensis (Groups A-C) and 2 related clades (Groups D & E) based on the analysis of 71 available internal transcribed spacer (ITS) sequences of O. sinensis in GenBank. To date, over 600 ITS sequences of O. sinensis have been registered in GenBank under Taxonomy ID: 72228, belonging to 17 genotypes (Li et al. 2016c, Zhu et al. 2016, Zhu & Li 2017). Genotype #2 of O. sinensis is a truncated form of Genotype #1 with 100% sequence homology but with different development/maturation patterns (Zhu et al. 2012). The other 15 mutants (Genotypes #3-#17) of O. sinensis contain numerous scattered transition, transversion, or insertion/deletion point

anamorph of this species is Hirsutella sinensis.” They

mutations or DNA segment reciprocal substitution

commented

in

hereditary variations between Genotype #1 H.

publications that suspiciously described this single

sinensis and a Group-E fungus (Figs. 1-3, Table 1)

fungal species as 3 independent genotype fungi.

(Stensrud et al. 2007, Yang et al. 2008, Xiao et al.

2B. Hypothesis II: O. sinensis is a collective term

2009, Zhang et al. 2009, Zhu et al. 2010, 2012, 2016,

for multiple fungi (Jiang & Yao 2002, Leung et al.

Yao et al. 2011, Li et al. 2016c, Zhu & Li 2017). Nine

2006, Zhu et al. 2007, Yang et al. 2008, Zhang et al.

genotypes (Genotypes #1-#3, #7-#12) are GC-biased;

2010, Barseghyan et al. 2011, Xia et al. 2015)

6 genotypes (Genotypes #4-#6, #15-#17) are AT-

on

the

“inaccurate”

wording

Jiang & Yao (2002) summarized the literature

biased; and Genotypes #13 & #14 are hereditary

findings of 22 fungal species spanning 13 genera that

variations with reciprocal substitution of large DNA

were identified from natural C. sinensis. Barseghyan

ZHU J-S et al. / Multiple genotypes of Ophiocordyceps sinensis

segments between Genotype #1 H. sinensis and the

2011, 2012, Li et al. 2016d), suggesting further

AB067719-type Group E O. sinensis fungus (cf. Fig.

exploration of additional genotypes of O. sinensis in

3). The sequences of mutant Genotypes #3-#17

natural C. sinensis. Albeit the considerable progress

belong to the genomes of independent fungi. Biochip-

in molecular studies, no studies to date have reported

based SNP mass spectrometry genotyping analysis

successful isolation, purification or morphological

identified

identification of Genotypes #2-#17 of O. sinensis.

additional

single-base

mutations

of

unknown sequences (Zhu et al. 2010, 2012, Gao et al.

3

4


Fig. 1 Alignment of ITS sequences of 8 O. sinensis genotypes with multiple scattered transition point mutations. AB067721 and HM595984 represent Genotypes #1 and #3 of GC-biased O. sinensis. AB067744 (Genotype #4), AB067740 (Genotype #5), EU555436 and KJ720572 (Genotype #6), KT232017 (Genotype #15), KT232019 (Genotype #16), and KT232010 (Genotype #17) represent 6 AT-biased genotypes of O. sinensis. Hyphens indicate identical bases, and spaces denote unmatched sequence gaps.


Fig. 2 Alignment of ITS sequences of O. sinensis genotypes with the multiple scattered transversion or insertion/deletion point mutations or DNA segment substitution genetic variations. AB067721 represents Genotype #1 H. sinensis, as the alignment reference. AJ488254 (reverse-complement), GU246286, GU246288, GU246287 and JQ695935 represent the GC-biased mutant Genotypes #7-#11 of O. sinensis. GU246280 represents Genotype #12, the insertion/deletion mutants of O. sinensis. KT339190 and KT339178 represent Genotypes #13 and #14, the DNA segment substitution genetic variations of O. sinensis. Hyphens indicate identical bases, and spaces denote unmatched sequence gaps. Table 1 Similarities between nrDNA sequences of 3 whole-genome H. sinensis vs. the sequences of multiple genotypes of O. sinensis.

Note: ANOV01021709 is the nrDNA segment of the whole-genome sequences ANOV00000000 for the H. sinensis strain Co18 (Hu et al. 2013). LKHE01000582 is the nrDNA segment of the whole-genome sequences LKHE00000000 for the H. sinensis strain 1229 (Li et al. 2016a). LWBQ01000008 is the nrDNA segment of the whole-genome sequences LWBQ00000000 for the H. sinensis strain ZJB12195 (Liu et

5

6


al. 2016). Sequences EF468971 (nrSSU) and EF468827 (nrLSU) were derived from the H. sinensis strain EFCC 7287, which was used as the O. sinensis standard in nomenclature studies (Sung et al. 2007, Quandt et al. 2014).

Fig. 3 Schematic illustration of the alignment of ITS1-5.8S-ITS2 sequences of Genotypes #1, #13, #14, and AB067719 of O. sinensis. AB067719 in lighter colors represents the sequences of the special Group E O. sinensis (Stensrud et al. 2007). AB067721 in darker colors represents the sequences of Genotype #1 H. sinensis. KT339178 and KT339190 represent the genetic variations, Genotypes #13 and #14 (offspring), of O. sinensis with reciprocal substitution of large DNA segments. Green bars represent the ITS1 segment; pink bars the 5.8S gene; and blue bars the ITS2 segment.

In contrast to most mutant genotypes, which

Genotypes #13 & #14 (Zhu et al. 2016). Regardless

exhibit multiple scattered point mutations, Genotypes

of whether the AB067719-type Group E fungus

#13 & #14 (offspring) of O. sinensis obtained from

belongs to O. sinensis (Taxonomy ID: 72228) as

the fully or semi-ejected ascospores of natural C.

classified by GenBank taxonomists or Fusarium sp.

sinensis represent hereditary variations, featuring

(Taxonomy

reciprocal substitutions of large DNA segments

1709257, 1709258), the discoveries of the ascosporic

between parental fungi, Genotype #1 H. sinensis and

Genotypes #13 & #14 along with the parental

a Group-E fungus represented by AB067719, as

ascosporic Genotype #1 and AB067719-type fungi

schematically illustrated in Fig. 3 (Zhu et al. 2016).

(Zhu et al. 2016) indicate possible nuclear fusion and

This AB067719 “O. sinensis” group includes at least

chromosomal interactions between the 2 parental

10

fungi

GenBank

entries

(AB067719,

AY986956,

ID:

during

981700,

the

1709218,

1709225,

development-maturation

and

AY986958, AY986959, LC163527, KT339197,

ejection of the teleomorphic ascospores of natural C.

KT340698, KX218226, KX218227, KX237742)

sinensis (cf. Fig. 3). The findings also suggest crucial

under Taxonomy ID: 72228. Sequences HM439576,

roles of these 2 parental fungi and their genetic

KJ735013, KJ735020, KJ735054, and KJ735055

offspring Genotypes #13 & #14 of O. sinensis in the

were identified from natural C. sinensis, sharing 99-

life cycle of natural C. sinensis, in particular,

100% sequence homology with AB067719, but

suggesting

labeled either as Fungal sp. (Taxonomy ID: 981700;

hyperparasitism in the combined consideration of the

Zhang et al. 2010) or Fusarium sp. (Taxonomy ID:

multicellular heterokaryotic ascospores with mono-

1709218, 1709225, 1709257, 1709258; Xia et al.

/bi-/tri-nucleate structure (Bushley et al. 2013).

possible

fungal

hybridization

or

2015). We identified 5 sequences from the ascospores

Three sets of whole-genome sequences for H.

of natural C. sinensis that were 99-100% homologous

sinensis have been documented: ANOV00000000 for

in sequence to AB067719 along with the discovered

the H. sinensis strain Co18 (Hu et al. 2013);


LKHE00000000 for the H. sinensis strain 1229 (Li et

in 8 of 15 strains isolated from the 25-day culture of

al. 2016a); and LWBQ00000000 for the H. sinensis

the single-ascospore of natural C. sinensis resulted in

strain ZJB12195 (Liu et al. 2016), the nrDNA

the debatable hypothesis of “ITS pseudogene”; but

sequences of which share 97.5-99.9% homology (cf.

the detection of only Genotype #1 in the remaining

Table 1). The GC-biased Genotype #1 (AB067721)

ascosporic strains and the lack of sequences of

shared 99.6-100% homology with the nrDNA

Genotypes #5 in the genome of the Genotype #1

segments of the H. sinensis whole-genome sequences

fungus contradict the “ITS pseudogene” hypothesis

(cf. Table 1). While Genotype #2 is excluded from

(Li et al. 2013). The genetic polymorphism of

Table 1 and Fig. 1 because it is a truncated form of

ascosporic

Genotype #1 with 100% sequence homology but with

multicellular heterokaryotic C. sinensis ascospores

distinct development-maturation pattern, the ITS

(parental) with mono-/bi-/tri-nucleate structures

sequences of Genotypes #3-#17 of O. sinensis

(Bushley et al. 2013, Li et al. 2013) was confirmed

showed low similarities (79-96%) with the whole-

by further culture-independent study of the C.

genome

in

sinensis ascospores with the detection of more

or

genotypes of O. sinensis (Zhu et al. 2016).

sequences.

ANOV00000000,

No

other

segments

LKHE00000000,

LWBQ00000000 displayed high similarities with any of the mutant O. sinensis Genotypes #3-#17. These O. sinensis

genotypes

(including

Genotype

#1)

differentially exist in the stroma, caterpillar body, ascocarps and ascospores of natural C. sinensis and the biomasses of the genotypes undergo dynamic alterations in an asynchronous fashion along with the maturation of natural C. sinensis (Xiao et al. 2009, Zhu et al. 2010, 2012, 2016, Gao et al. 2011, 2012, Yao et al. 2011, Li et al. 2016b, c, Zhu & Li 2017). All these findings indicate that the mutant genotypes reside not in the genome of Genotype #1 H. sinensis but in the genomes of independent O. sinensis fungi (Xiao et al. 2009, Zhu et al. 2010, 2012, 2016, Li et al. 2016b, Zhu & Li 2017), which is inconsistent with the sole anamorph hypothesis for H. sinensis (Hypothesis I) (Wei et al. 2006, Guo et al. 2010) but consistent

with

Hypothesis

III,

the

multiple

independent mutant O. sinensis fungi hypothesis (Xiao et al. 2009, Zhang et al. 2009, Zhu et al. 2010,

strains

(offspring)

derived

from

Zhang et al. (2009) analyzed 56 C. sinensis specimens collected from the Tibet, Qinghai, Sichuan and Yunnan provinces and reported genetic variations in O. sinensis. ITS sequences (FJ654148 and FJ654149) derived from 2 of 11 Nyingchi (Tibet) isolates belong to GC-biased Genotype #3 (Figs. 1,4, Table 1), while ITS sequences of the rest isolates from Nyingchi or other production areas belong to Genotype #1 H. sinensis. Accordingly, the authors speculated that Nyingchi of Tibet was the center of origin of O. sinensis. Regardless of the validity of the hypotheses

of

“the

center

of

origin”

and

phylogeographic evolution for O. sinensis, in particular when questioning whether the GC-biased Genotype #1 or #3 coexists with AT-biased genotypes of O. sinensis in natural C. sinensis, this study did confirm the genetic diversity of O. sinensis and that the sequences of Genotype #3 reside not in the genome of Genotype #1 H. sinensis.

2012, 2016, Gao et al. 2011, 2012, Yao et al. 2011, Li

In contrast to the geographic difference in O.

et al. 2016b, Zhu & Li 2017) and the “integrated

sinensis genotypes reported by Zhang et al. (2009),

micro-ecosystem” hypothesis for natural C. sinensis

Mao et al. (2013) reported a different geographic

(Liang et al. 2010).

distribution of Genotypes #4 and #5 of O. sinensis

Although the simultaneous detection of both

detected from C. sinensis specimens collected from

GC-biased Genotype #1 and AT-biased Genotype #5

different production areas in the Tibet, Sichuan, Qianghai, and Yunnan provinces but did not detect

7

8


Genotype #1 H. sinensis in the samples. Genotypes

H. sinensis, and the conidia were larger for Genotype

#4 and #5 of O. sinensis were observed sharing the

#4 than for Genotype #5.

same microscopic morphology as that of Genotype #1

Fig. 4 Phylogenetic relationships between mutant genotypes of O. sinensis fungi. Three nrDNA sequences of the whole-genome of H. sinensis and 80 O. sinensis ITS sequences under GenBank Taxonomy ID: 72228 (except


the first-registered Genotype #6 sequence EU555436 under Taxonomy ID 175245) entered into the phylogenetic analysis using the Neighbor Joining algorithm. Genotype #1 sequences are with no labels. Genotype #2 is excluded from this analysis due to having 100% sequence homology with Genotype #1 (Zhu et al. 2012). The GC-biased Genotypes #3 and #7-#12 are labeled in blue, and the AT-biased Genotypes #4-#6 and #15-#17 are labeled in red. Those labeled in green indicate a special O. sinensis cluster containing Genotypes #13-#14 of O. sinensis, the genetic variations (offspring) with reciprocal substitutions of large DNA segments between the 2 parental O. sinensis fungi, Genotype #1 H. sinensis and an AB067719-type fungus (Group E O. sinensis) (cf. Fig. 3). ANOV01021709 (=KE659721) is the nrDNA segment of the whole-genome sequences (ANOV00000000) for the H. sinensis strain Co18 (Hu et al. 2013); LKHE01000582 is the nrDNA segment of the whole-genome sequences (LKHE00000000) for the H. sinensis strain 1229 (Li et al. 2016a); and LWBQ01000008 is the nrDNA segment of the whole-genome sequences (LWBQ00000000) for the H. sinensis strain ZJB12195 (Liu et al. 2016).

Wei et al. (2006) assumed that the teleomorph of

fungal

(species)

complex

containing

several

O. sinensis was GC-biased Genotype #1 fungus; the

genotypes of O. sinensis and Paecilomyces hepiali

key authors of the paper reported 10 years later that

(Li et al. 2016d), or that secondary infections, if not

the teleomorph of O. sinensis was AT-biased

primary, occurred during artificial cultivation. If

Genotype #4 fungus, distinct from the sole

neither of these 2 possibilities is scientifically

anamorphic Genotype #1 H. sinensis that was

supported, a preprogrammed, non-random mutagenic

detected in 3 inoculation agents used for artificial

conversion of GC-biased Genotype #1 to AT-biased

cultivation (Wei et al. 2016). As described above, the

Genotype #4 may have occurred unexceptionally in

sequences of Genotype #4 were not detected in the

all individual pieces of artificial C. sinensis during

genome of Genotype #1 H. sinensis and were not

cultivation, although this seems very unlikely.

present in the ascospores of natural C. sinensis (Li et

The above review indicates that O. sinensis is

al. 2013, Zhu et al. 2016). Phylogenetic analysis

not a single fungus (Hypothesis I) but rather a

placed the GC-biased Genotype #1 H. sinensis and

collective name for multiple fungi (Hypotheses II &

the AT-biased Genotype #4 in far isolated clades in

III), and the multiple mutant genotypes represent O.

the phylogenetic tree (Fig. 4). These findings

sinensis fungi independent of Genotype #1 H.

advocate against the implementation of 1F=1N in C.

sinensis (Xiao et al. 2009, Zhu et al. 2010, 2012,

sinensis studies by providing solid evidence that

2016, Gao et al. 2011, 2012, Yao et al. 2011, Li et al.

supplements the available evidence in the literature

2016b, c, Zhu & Li 2017). These results support

and strongly endorsing Hypothesis III, which holds

adherence to the 1F=1N rule as long as the following

that O. sinensis is a collective name for multiple

findings are validated: (1) O. sinensis is a single

mutant genotypes of O. sinensis fungi (Zhu & Wu

fungus, and (2) H. sinensis is the sole anamorph of O.

2015, Li et al. 2016c, Yao & Zhu 2016, Zhu & Li

sinensis (Hawksworth et al. 2011, McNeill et al.

2017). The apparent mystery of “planting melon

2012, Yao & Zhu 2016).

seeds and harvesting beans” may imply that the sequences of Genotype #4 have been overlooked among

the

inoculation

strains

in

combined

consideration that the actual causal agent may be a

3 HISTORY OF THE INDISCRIMINATE USE OF THE SAME LATIN NAME FOR THE NATURAL INSECT-FUNGI COMPLEX AND FOR TELEOMORPHIC AND ANAMORPHIC O.

9

10


meaning of the name and led to confusion in C.

SINENSIS FUNGI Although the proposal of implementing 1F=1N in C. sinensis research (Zhang et al. 2013b) did not

sinensis research (Zhang et al. 2012, Ren et al. 2013, Li et al. 2016c, Yao & Zhu 2016, Zhu & Li 2017).

initiate the indiscriminate use of the same Latin name

This “herbal” medicine was originally named

in C. sinensis studies, it did contribute to further

DōngChóngXìaCăo 冬虫夏草 , also known as

deterioration during the long history of this

XìaCăoDōngChóng 夏草冬虫 (HiaTsaoTomChom,

indiscriminate practice (Li et al. 2016c, Yao & Zhu

HiaTsaoTomTchom,

2016, Zhu & Li 2017). The term “O. sinensis” now refers to the natural insect-fungi complex, the

HeaTsaonTsongChung), DōngChóngCăo 冬虫草 , ChóngCăo 虫草 (TchongTsao) and དབྱར་རྩྭ་དགུན་འབུ,

teleomorphic O. sinensis fungi, and the postulated

Yartsa Gunbu and Yarchagumba, Totsu Kaso and

anamorphic H. sinensis, Tolypocladium sinensis

Tochukaso in different languages. (Winkler 2008,

(Barseghyan et al. 2011), and multiple mutant

Panda & Swain 2011, Shrestha et al. 2013, Lu 2014,

genotypes of O. sinensis fungi.

Yao & Zhu 2016). The first record of this folk

HiaTsaoTongTchong,

Natural C. sinensis is one of the most valued

medicine appears in "Man ngag bye ba ring bsrel",

“herbs” in traditional Chinese medicine (TCM) with

written by the Tibetan doctor Zur Mkhar Mnyam

a rich history of traditional use as a folk medicine for

Nyid Rdo Rje (1439-1475) (Pegler et al. 1994,

health maintenance, disease amelioration, post-

Xizang Institute of Tibetan Hospital and Medicine

disease recovery, and anti-aging therapy (Zhu et al.

2002, Winkler 2008, Lu 2014, Yao & Zhu 2016).

1998a, b, Li et al. 2011, Tan et al. 2011). Natural C.

After a centuries-long history of clinical use,

sinensis consists of the fruiting body of the multiple

DōngChóngXìaCăo was recorded in ancient TCM

O. sinensis fungi and a dead larva of a Hepialidae

books in Mandarin, including “Essentials of Materia

moth as an insect-fungi complex, therefore natural C.

Medica 本草备要” by Wang Ong (1694), “Light of

sinensis ≠ O. sinensis fungi (Jiang & Yao 2002, Zhu

Embers for Materia Medica 青藜馀照 ” by Tang

et al. 2007, Zhang et al. 2012, Bushley et al. 2013,

Fangyi (1712-1722), “New Compilation of Materia

Ren et al. 2013, Xia et al. 2015, Zhu & Wu 2015, Li

Medica 本草从新 ” by Wu Yiluo (1757), and “A

et al. 2016b, Yao & Zhu 2016, Zhu & Li 2017).

Supplement to the Compendium of Materia Medica

However, the same Latin name, C. sinensis or O.

本草纲目拾遗” by Zhao Xuemin (1765) (Lu 2014).

sinensis, has been applied indiscriminately to the C. sinensis host-fungi complex and

O.

sinensis

Natural C. sinensis was first introduced to Western countries by the

French

missionary

fungus/fungi (Lindley 1846, Berkeley 1857, Pratt

Dominicus Parennin in 1723 (Lu 2014). The intrinsic

1892, Massee 1895, Lloyd 1918, Jiang & Yao 2002,

fungus was identified as belonging to the Sphaeria

Chen et al. 2004, Leung et al. 2006, Wei et al. 2006,

genus by Jonathan Pereira in 1843; the insect portion

2016, Stensrud et al. 2007, Guo et al. 2010, Liang et

was identified as belonging to Agrotis according to

al. 2010, Shrestha et al. 2010, 2013, Barseghyan et al.

Edward Doubleday (Saunders 1842, Pereira 1843, Lu

2011, Bushley et al. 2013, Hu et al. 2013, Lo et al.

2014). Miles Joseph Berkeley described the fungus as

2013, Lu 2014, Zhang et al. 2013a, Quandt et al.

Sphaeria Sinensis Berkeley in 1843 and renamed it

2014, Xiang et al. 2014, Zhou et al. 2014, Xia et al.

Cordyceps Sinensis in 1857 (Berkeley 1843, 1857, Lu

2015, Li et al. 2016a); this has obscured the specific

2014, Yao & Zhu 2016). Pier Andrea Saccardo placed


this species in the genus Cordyceps (Ft.) Link in 1878

al. 2012, Ren et al. 2013) and have proposed

and renamed it Cordyceps sinensis (Berkeley)

implementing IMA 1F=1N in C. sinensis research and

Saccardo in 1883 (Saccardo 1878, 1883, Lu 2014).

replacing H. sinensis with the teleomorphic name O.

After a century of use, Cordyceps sinensis was

sinensis (Zhang et al. 2013b). Quandt et al. (2014)

renamed Ophiocordyceps sinensis (Berkeley) G.H.

proposed suppressing the use of Hirsutella and

Sung et al. (2007), when fungal strain EFCC 7287

merging Hirsutella under Ophiocordyceps based on

was arbitrarily selected as a nomenclature standard

their findings using strain EFCC 7287 (H. sinensis)

(according to Dr. Hywel-Jones in a ResearchGate

and strains of other Hirsutella species as standards.

discussion).

The H. sinensis strain EFCC 7287 was arbitrarily

Derived from the strain EFCC 7287 (Sung et al.

selected as the standard for O. sinensis by Sung et al.

2007, Quandt et al. 2014), the nrLSU (the large

(2007) (according to Dr. Hywel-Jones). As a result,

subunit of nuclear ribosomal DNA) sequence

O. sinensis (≡C. sinensis) is indiscriminately used to

(EF468827) is 99.5-99.7% homologous to the nrDNA

refer not only to the insect-fungi complex and to

segments of the whole-genome sequences (Table 1):

teleomorphic heterokaryotic fungi but also to the

ANOV01021709 in ANOV00000000 for the strain

postulated anamorph of O. sinensis, the homokaryotic

Co18 (Hu et al. 2013), LKHE01000582 in

fungus H. sinensis (Bushley et al. 2013, Li et al. 2013,

LKHE00000000 for the strain 1229 (Li et al. 2016a),

2016c, Mao et al. 2013, Zhang et al. 2013a, Yao &

and LWBQ01000008 in LWBQ00000000 for the

Zhu 2016, Zhu & Li 2017).

strain ZJB12195 (Liu et al. 2016); the nrSSU (the

Blurring the differences between the natural

small subunit of nuclear ribosomal DNA) sequence

insect-fungi

(EF468971) is 98.7-99.5% homologous to segments

anamorphic O. sinensis fungi unavoidably leads to

of LKHE01000582 and LWBQ01000008 (Li et al.

confusion. For example, Hu et al. (2013) disclosed

2016a, Liu et al. 2016). These sequence analyses

that “O. sinensis strain Co18 … was selected for

indicate that the renaming of C. sinensis to O. sinensis

genome sequencing” but also wrote that “The

by Sung et al. (2007) was only for Genotype #1 H.

caterpillar

sinensis,

without

legitimately

extending

complex

fungus

and

teleomorphic

Ophiocordyceps

and

sinensis

their

(…anamorph: Hirsutella sinensis) is one of the most

nomenclature project to the naturally coexistence of

highly valued traditional Chinese medicines”. They

diverse O. sinensis genotypes that belong to the

use the term “O. sinensis” indiscriminately to refer to

genomes of independent fungi (cf. Figs. 1-4, Table 1).

the teleomorphic and anamorphic fungus and the

Given the definition of natural C. sinensis ≠ O.

natural insect-fungi complex. The corresponding

sinensis fungi by the Chinese Pharmacopeia, botany-

author of (Hu et al. 2013) explained to the audience

TCM systematics describes C. sinensis as the entire

at an international medicinal mycology conference

insect-fungi complex; the renaming of C. sinensis to

that the study material was an anamorphic H. sinensis

O. sinensis (Sung et al. 2007) was related only to the

Co18 strain purified through the single-conidia

fungus/fungi and did not impact the name of the

approach. Regardless of how clearly he made his case

natural insect-fungi complex. Mycologists state that

to a small audience at that conference, his paper (Hu

O. sinensis (≡C. sinensis) refers to the fungus/fungi

et al. 2013) has been distributed globally with

that form part of the insect-fungi complex (Zhang et

corresponding

confusion

regarding

the

study

11

12


specimen. Because of this confusion, Zhang & Zhang

the genomes of other fungi (Zhu & Wu 2015, Li et al.

(2015) and Zhang et al. (2016) cited (Hu et al. 2013)

2016c, Zhu & Li 2017). Although the teleomorphic

and concluded that genome sequencing of O. sinensis

hypothesis for GC-biased Genotype #1 O. sinensis

has been completed. Clearly, these conclusions

encourages the implementation of 1F=1N in C.

overlooked important differences between the

sinensis research (Wei et al. 2006, Zhang et al.

homokaryotic anamorphic H. sinensis strain Co18

2013b),

that was labeled O. sinensis in the cited study (Hu et

implementation: the detections of the teleomorphic

al. 2013) and the teleomorphic heterokaryons with

AT-biased Genotype #4 of O. sinensis in artificial C.

multicellular mono-, bi- and tri-nucleated structures

sinensis and of Genotypes #3-#5 with no detection of

and enormous genetic heterogeneity of O. sinensis

Genotype #1 H. sinensis in natural C. sinensis (Zhang

that were used in many other studies (Bushley et al.

et al. 2009, Mao et al. 2013, Wei et al. 2016) and of

2013, Li et al. 2013, 2016b, c, Zhu et al. 2016, Zhu &

Genotypes #5-#6, #13-#14, and #16 of O. sinensis in

Li 2017). The conclusions also overlooked the fact

the ascospores of natural C. sinensis (Li et al. 2013,

that multiple fungi, including H. sinensis, have been

Zhu et al. 2016).

other

findings

advocate

against

the

postulated as the anamorphs of O. sinensis (Jiang &

There are many other examples of confusing

Yao 2002, Barseghyan et al. 2011) and the genetic

statements in C. sinensis studies. For instance, Zhang

variant offspring Genotypes #13 and #14 of O.

et al. (2009) stated that “Ophiocordyceps sinensis …

sinensis detected from the ascospores of natural C.

has been widely used in traditional Chinese medicine

sinensis (Zhu et al. 2016), which feature the

for the treatment of asthma, bronchial, lung

reciprocal substitutions of large DNA segments

inflammation … O. sinensis infects soil-borne larvae

between their parental fungi, H. sinensis and an

of more than 50 species of ghost moths”, while the

AB067719-type, Group-E O. sinensis fungus (cf.

authors identified either Genotype #1 or #3 (not both)

Figs. 3-4, Table 1). Due to the genetic diversity and

from C. sinensis specimens collected from different

morphological differences between anamorphic and

geographic areas. The title of (Liu et al. 2015),

teleomorphic O. sinensis fungi, general statements

“Transcriptome sequencing and analysis of the

indicating

for

entomopathogenic fungus Hirsutella sinensis isolated

Ophiocordyceps sinensis has been completed”

that

“the

genome

sequencing

from Ophiocordyceps sinensis”, represents another

(Zhang & Zhang 2015) or “Chinese medicinal fungi

example in which O. sinensis obviously refers to the

(Ophiocordyceps sinensis …) have been genome

insect-fungi complex; here, replacement of the name

sequenced” (Zhang et al. 2016) are scientifically

H. sinensis by O. sinensis will result in the chaotic

inaccurate and can further confuse readers.

logic of “O. sinensis isolated from O. sinensis”.

Three strains of H. sinensis (all labeled as O.

Barseghyan et al. (2011) concluded that “the

sinensis) were used for whole-genome sequencing

investigated strains were identified as Hirsutella

(Hu et al. 2013, Li et al. 2016a, Liu et al. 2016).

sinensis and Tolypocladium sinensis species, which

Further analyses against the H. sinensis whole-

were identified as anamorphs of Ophiocordyceps

genome sequences indicated that the sequences of

sinensis”; here, the anamorphic names H. sinensis and

multiple mutant genotypes (Genotypes #2-#17) of O.

T. sinensis cannot be replaced by O. sinensis.

sinensis reside not in the genome of H. sinensis but in

In aggregation, there are multiple mutant O.


sinensis fungi, namely, GC-biased Genotypes #1-#3

and the causal fungus/fungi. However, this suggestion

and #7-#12, AT-biased Genotypes #4-#6 and #15-

may be controversial because the taxonomic positions

#17, and hereditary variations Genotypes #13 and #14

of Genotypes #2-#17 of O. sinensis fungi described

with reciprocal substitution of large DNA segments

previously are still morphologically undetermined

between Genotype #1 H. sinensis and the AB067719-

due to lack of success in culturing, purifying and

type Group E O. sinensis fungus, whose sequences

identifying these mutant organisms (Zhang et al.

belong to the genomes of independent fungi (cf. Figs.

2013a, Zhu & Wu 2015, Li et al. 2016c, Zhu et al.

1-4, Table 1). The aforementioned indiscriminate use

2016,

Zhu

&

Li

2017)

and

due

to

the

th

indistinguishable H. sinensis morphology shared by

century when Western scholars began to identify

Genotypes #1 H. sinensis, #4 and #5 of O. sinensis

intrinsic fungal species with the intention of linking

fungi (Mao et al. 2013) and the “Hirsutella-like”

the well-regarded TCM regime with a fungus. This

morphology produced by the numerous fungal

situation deteriorated following implementation of

species (Quandt et al. 2014). It remains unclear

the 1F=1N rule in C. sinensis research.

whether the mutant fungi are variants within the

of the same Latin name began in the middle of the 19

4 RENAMING SUGGESTIONS NATURAL C. SINENSIS

FOR

Based on the definition of natural C. sinensis ≠ O. sinensis fungi by the Chinese Pharmacopeia, a group of mycologists suggested continuing to use the term O. sinensis for the fungus/fungi and renaming the natural product using the non-Latin term “Chinese cordyceps” (Zhang et al. 2012). This proposal was not

generally

accepted

because

governmental

regulations worldwide require every natural product to have an exclusive Latin name. Questioning the necessity of an additional non-Latin name for natural C. sinensis when a number of non-Latin names have already been used for centuries, many authors have directly used the Chinese phonetic alphabet name DōngChóngXìaCăo and/or its abbreviation DCXC, or the Tibetan alias Yartsa Gunbu or Yarchagumba (Winkler 2008, Panda & Swain 2011, Lo et al. 2013, Zhao et al. 2013, 2015, Dworecka-Kaszak 2014, Zhou et al. 2014, Pradhan 2016). In contrast, other mycologists suggested the Latin name “Ophiocordyceps & Hepialidae” for natural C. sinensis (Ren et al. 2013), reflecting the parasitic relationship between the larva of bat moths

genus Ophiocordyceps Petch. Stensrud et al. (2007) believed “a large sequence variation of the 5.8S nrDNA far exceeds what is normally observed in fungi … even at higher taxonomic levels (genera and family)” and hypothesized that C. sinensis includes multiple cryptic species. Other mycologists have argued against this hypothesis (Xiao et al. 2009, Liang et al. 2010, Li et al. 2013, Zhang et al. 2013a), some of them hypothesized that these mutant genotypes represent independent fungal species, rather than cryptic species. Given the identification of both H. sinensis and T. sinensis as anamorphs of O. sinensis (Barseghyan et al. 2011), the proposal “for Ophiocordycipitaceae

(Hypocreales)

with

new

combinations in Tolypocladium” (Qaundt et al. 2014) and the assumption of multiple mutant genotypes of O. sinensis in that “a large sequence variation of the 5.8S nrDNA far exceeds what is normally observed in fungi”

but

probably

within

the

Family

Ophiocordycipitaceae (Stensrud et al. 2007), it may be

reasonable

to

revise

the

proposed

term

“Ophiocordyceps & Hepialidae” for the natural C. sinensis insect-fungi complex (Ren et al. 2013) to the new term “Ophiocordycipitaceae & Hepialidae” for

13

14


the nomenclatural consideration of both the dead

sinensis have been demonstrated residing not in the

larva of the Family Hepialidae and the multiple

genome of Genotype #1 H. sinensis, but rather in the

anamorphs and teleomorphs of O. sinensis of the

genomes of independent fungi. The mutant O.

Family Ophiocordycipitaceae, including multiple

sinensis fungi differentially exist in the different

mutant genotypes of O. sinensis and other fungal

compartments of natural C. sinensis and exhibit the

species, such as Tolypocladium sinensis as one of the

development-maturation patterns distinct from that of

anamorphs of O. sinensis (Barseghyan et al. 2011,

Genotype #1 H. sinensis. In this situation, the forced

Qaundt et al. 2014). However, special taxonomic

implementation of 1F=1N has expanded the

attention is needed in the case of the genetic variant

centuries-long conventional, albeit indiscriminate,

offspring Genotypes #13 and #14 of O. sinensis (Zhu

use of the same Latin name not only for the natural C.

et al. 2016), which feature reciprocal substitutions of

sinensis insect-fungi complex but also for multiple

large DNA segments between their parental fungi, H.

anamorphs and teleomorphs of O. sinensis fungi. This

sinensis (AB067721) and a Group E O. sinensis

deterioration has led to problems in academia and has

fungus (AB067719) (cf. Fig. 3). If Group E O.

negatively impacted the formation and execution of

sinensis fungus (AB067719) is proven to belong to

governmental policies and the market psychology of

Fusarium sp. (Zhang et al. 2010, Xia et al. 2015), the

consumers and the general population. We therefore

ascosporic Genotypes #13 and #14 of O. sinensis may

propose the following resolution of the nomenclatural

represent the outcomes (offspring) of chromosomal

problems. We suggest (1) a revised Latin name,

(nuclear) fusion and reciprocal substitutions of large

“Ophiocordycipitaceae & Hepialidae”, for the

DNA segments of the parental fungi, possibly

natural

suggesting biological courses of hyperparasitism or

distinguish between the natural insect-fungi complex

mating types other than homothallic or pseudo-

and the O. sinensis fungi (teleomorphs and

homothallic, in combined consideration of the

anamorphs); (2) postponing the implementation of

multicellular heterokaryotic ascospores of natural C.

1F=1N in C. sinensis studies until it has been

sinensis with the mono-/bi-/tri-nucleate structures

demonstrated that O. sinensis is indeed a single

and the genetic heterogeneity of the C. sinensis

fungus and that H. sinensis is indeed the sole

ascospores (Bushley et al. 2013, Li et al. 2013,

anamorph of O. sinensis; (3) postponing the

2016b, Zhu et al. 2016).

execution of the proposal of merging Hirsutella under

C.

sinensis

insect-fungi

complex

to

Although several renaming proposals have been

Ophiocordyceps (this can be re-proposed after their

raised for the C. sinensis insect-fungi complex, the

study is expanded to include the multiple mutant

nomenclature for natural C. sinensis and O. sinensis

genotypes of O. sinensis); and (4) encouraging

fungi

taxonomists

is

highly

academic

and

requires

the

worldwide

to

join

efforts

in

considerations of many other factors and the full

characterizing the multiple mutant genotype fungi

cooperation of multidisciplinary taxonomists across

that are currently grouped under the name O. sinensis.

the academic fields of mycology, zoology and

These proposals seek solutions to the long-lasting

botany-TCM.

academic problem of indiscriminate use of the same

5 CONCLUSIONS AND SUGGESTIONS The multiple Genotypes #2-#17 of mutant O.

Latin name for the natural insect-fungi complex and for teleomorphs and anamorphs of O. sinensis fungi


to avoid compromising scientific understanding and to alleviate the social consequences that arise from

Xue Ban 43: 259–266. Guo Y-L, Xiao P-G, Wei J-C, 2010.. On the biology and sustainable

problems in nomenclature.

utilization of the Chinese medicine treasure Ophiocordyceps

[REFERENCES]

sinensis (Berk.) G. H. Sung et al. Modern Chinese Medicine

Barseghyan GS, Holliday JC, Price TC, Madison LM, Wasser SP, 2011. Growth and cultural-morphological characteristics of vegetative

mycelia

of

medicinal

caterpillar

fungus

Ophiocordyceps sinensis GH Sung et al. (Ascomycetes) isolates from Tibetan Plateau (P.R.China). International

Hawksworth DL, Crous PW, Redhead SA, Reynolds DR, Samson RA, Seifert KA, Taylor JW, Wingfield MJ, Abaci O, Aime C, Asan A, Bai F-Y, de Beer ZW, Begerow D, Berikten D, Boekhout T, Buchanan PK, Burgess T, Buzina W, Cai L, Cannon PF, Crane JL, Damm U, Daniel HM, van Diepeningen

Journal of Medicinal Mushrooms 13: 565–581. Berkeley MJ, 1843. On some entomogenous Sphaeriae. London

AD, Druzhinina I, Dyer PS, Eberhardt U, Fell JW, Frisvad JC, Geiser DM, Geml J, Glienke C, Gräfenhan T, Groenewald JZ,

Journal of Botany 2: 205–211. Berkeley MJ, 1857. On some Entomogenous Sphaeriae. Journal of the Proceedings of the Linnean Society of London. Botany 1:

Groenewald M, de Gruyter J, Guého-Kellermann E, Guo L-D, Hibbett DS, 2011. The Amsterdam Declaration on fungal nomenclature. IMA Fungus 2: 105–112.

157–159. doi:10.1111/j.1095-8339.1857.tb02440.x. Bushley KE, Li Y, Wang W-J, Wang X-L, Jiao L, Spatafora JW, Yao Y-J, 2013. Isolation of the MAT1-1 mating type idiomorph and evidence for selfing in the Chinese medicinal fungus Ophiocordyceps sinensis. Fungal Biology 117: 599–

He S-Q, Jin X-L, Lou J-C, Wang C-M, 2011. Morphological characteristics and submerged culture medium screening of Pseudogymnoascus roseus. Microbiology, (China) 38: 1371– 1376. He S-Q, Jin X-L, Lou J-C, Wang C-M, Wang S-R, 2010.

610. Chen Y-Q, Hu B, Xu F, Zhang W-M, Zhou H, Qu L-H, 2004. Genetic variation of Cordyceps sinensis, a fruit-bodyproducing

12: 3–8.

entomopathogenic

species

from

different

geographical regions in China. FEMS Microbiology Letters 230: 153–158. Dworecka-Kaszak B, 2014. Cordyceps fungi as natural killers, new hopes for medicine and biological control factors. Annals of Parasitology 60: 151–158. Gao L, Li X-H, Zhao J-Q, Lu J-H, Zhao J-G, Zhu J-S, 2012. [Maturation of Cordyceps sinensis associates with alterations of fungal expressions of multiple Ophiocordyceps sinensis mutants in stroma of Cordyceps sinensis]. Beijing da Xue Xue Bao. Yi Xue Ban 44: 454–463. Gao L, Li X-H, Zhao J-Q, Lu J-H, Zhu J-S, 2011. Detection of multiple Ophiocordyceps sinensis mutants in the stroma of premature Cordyceps sinensis by MassARRAY SNP MALDITOF mass spectrum genotyping. Beijing da Xue Xue Bao. Yi

Morphological characteristics and submerged culture medium screening of a strain of Geomyces pannorum isolated from Cordyceps sinensis. Chinese Journal of Grassland 32: 70–75. Hu X, Zhang Y-J, Xiao G-H, Zheng P, Xia Y-L, Zhang X, St Leger RJ, Liu X-Z, Wang C-S, 2013. Genome survey uncovers the secrets of sex and lifestyle in caterpillar fungus. Chinese Science Bulletin 58: 2846–2854. Jiang Y, Yao Y-J, 2002. Names related to Cordyceps sinensis anamorph. Mycotaxon 84: 245–254. Jiang Y, Yao Y-J, 2005. ITS sequence analysis and ascoma development of Pseudogymnoascus roseus. Mycotaxon 94: 55–73. Kai Z, Yongjian L, Sheng G, Yu L, 2015. Effect of Dongchongxiacao (Cordyceps) therapy on contrast-induced nephropathy in patients with type 2 diabetes and renal insufficiency undergoing coronary angiography. Journal of Traditional Chinese Medicine = Chung i Tsa Chih Ying Wen

15

16


Pan 35: 422–427.

Research 14: 96–112.

Leung PH, Zhang Q-X, Wu J-Y, 2006. Mycelium cultivation, chemical

composition

and

antitumour

activity

of

a

Tolypocladium sp. fungus isolated from wild Cordyceps sinensis. Journal of Applied Microbiology 101: 275–283. Li C-L, Tan N-Z, Barger JL, Zhang Y, Ferguson SB, Prolla TA,

Liang Z-Q, Han Y-F, Liang J-D, Dong X, Du W, 2010. Issues of concern

in

the

studies

of

Ophiocordyceps

sinensis.

Microbiology, (China) 37: 1692–1697. Lindley J, 1846. The vegetable kingdom, vol. 39. Bradbury & Evans, London.

Zhu J-S, 2011. Combined use of whole-gene expression

Liu X-J, Guo Y-L, Yu Y-X, Zeng W, 1989. Isolation and

profiling technology and mouse lifespan test in anti-aging

identification of the anamorph of Cordyceps sinensis fungus.

herbal product study Proceedings of New TCM Products

Acta Mycologica, (Sinica) 8: 35–40.

Innovation and Industrial Development Summit. China

Liu Z-Q, Lin S, Baker PJ, Wu L-F, Wang X-R, Wu H, Xu F, Wang

Journal Chinese Materia Medica and Zhejiang: 443–448.

H-Y, Brathwaite ME, Zheng Y-G, 2015. Transcriptome

University Publishing, Hangzhou.

sequencing and analysis of the entomopathogenic fungus

Li Y, Jiao L, Yao Y-J, 2013. Non-concerted ITS evolution in fungi, as

revealed

from

the

important

medicinal

fungus

Ophiocordyceps sinensis. Molecular Phylogenetics and Evolution 68: 373–379.

Yao Y-J, 2016a. Comparison of different sequencing and assembly strategies for a repeat-rich fungal genome, sinensis.

BMC Genomics 16: 106. Liu Z-Q, Lin S, Zheng Y-G, 2016. Genome sequencing and analysis of anamorphic strain (Hirsutella sinensis) from traditional

Li Y, Yang R-H, Hu X-D, Wang K, Wang W-J, Wang X-L, Jiao L,

Ophiocordyceps

Hirsutella sinensis isolated from Ophiocordyceps sinensis.

Journal

of

Microbiological

Methods: 128: 1-6. Li Y-L, Yao Y-S, Wu Z-M, Chen X, Zhang Z-H, Liu X, Xu H-F, Ma S-L, Xie W-D, Wu J-Y, Zhu J-S, 2016b. Co-existence of multiple mutant genotypes of Ophiocordyceps sinensis fungi

medicine

Ophiocordyceps

sinensis.

https://www.ncbi.nlm.nih.gov/nuccore/1031242549. Liu Z-Y, Yao Y-J, Liang Z-Q, Liu A-Y, Pegler DN, Chase MW, 2001. Molecular evidence for the anamorph-teleomorph connection in Cordyceps sinensis. Mycological Research 105: 827-832. Lloyd CG, 1918. Cordyceps sinensis, From N. Gist Gee, China. Mycology Notes 54: 766–780.

in the two types of the Cordyceps sinensis ascospores forming

Lo H-C, Hsieh C, Lin F-Y, Hsu T-H, 2013. A systematic review of

multicellular heterokaryons Proceedings of 2016 The 11th

the mysterious caterpillar fungus Ophiocordyceps sinensis in

National Conference of Medicinal Mycology. November 28:

Dong-Chong-Xia-Cao and related bioactive ingredients.

50–59. Chengdu, Sichuan, China.

Journal of Traditional and Complementary Medicine 3: 16-32.

Li Y-L, Yao Y-S, Xie W-D, Zhu J-S, 2016c. The Molecular

Lu D, 2014. Western records and studies of the Chinese caterpillar

heterogeneity of natural Cordyceps sinensis with multiple

fungus to the beginning of the 20th Century. Journal of Fungal

Ophiocordyceps sinensis fungi challenges the anamorph-

Research 12: 233–244.

teleomorph connection hypotheses. American Journal of Biomedical Sciences 8: 123–159. Li Y-L, Yao Y-S, Zhang Z-H, Xu H-F, Liu X, Ma S-L, Wu Z-M, Zhu J-S, 2016d. Synergy of fungal complexes isolated from the intestines of Hepialus armoricanus larvae containing multiple fungi increases infection potency. Journal of Fungal

Mao X-M, Zhao S-M, Cao L, Yan X, Han R-X, 2013. The Morphology observation of Ophiocordyceps sinensis from different origins. Journal of Environmental Entomology 35: 343–353. Massee G, 1895. A revision of the genus Cordyceps. Annals of Botany os–9: 1–44.


McNeill J, Barrie FR, Buck WR, Demoulin V, Greuter W, Hawksworth DL, Herendeen PS, Knapp S, Marhold K, Prado J, Prud’Homme van Reine WF, Smith GF, Wiersema JH,

entomological society. Annals and Magazine of Natural History 8: 217. Shrestha B, Zhang W-M, Zhang Y-J, Liu X-Z, 2010. What is the

Turland NJ, 2012. International code of nomenclature for

Chinese

algae, fungi, and plants (Melbourne Code). Regnum

(Ophiocordycipitaceae)? Mycology 1: 228–236.

Vegetabile 154. A.R.G. Koeltz Scientific Books, Königstein, Germany.

caterpillar

fungus

Ophiocordyceps

sinensis

Shrestha S, Shrestha AK, Park JH, Lee DY, Cho JG, Shrestha B, Baek NI, 2013. Review on pharmacologically active

Panda AK, Swain KC, 2011. Traditional uses and medicinal

metabolites from Yarsagumba (Ophiocordyceps sinensis), an

potential of Cordyceps sinensis of Sikkim. Journal of

epitome of Himalayan elixir. Nepal Journal of Science and

Ayurveda and Integrative Medicine 2: 9–13.

Technology 14: 49–58.

Pegler DN, Yao Y-J, Li Y (1994) The Chinese ‘caterpillar fungus’. Mycologist 8: 3–5.

Kauserud H, 2007. Accelerated nrDNA evolution and

Pereira J, 1843. Notice of a Chinese article of the materia medica, called “summer-plant-winter-worm”. Pharmaceutical Journal and Transactions 2: 591–595.

(Ascomycetes):

profound AT bias in the medicinal fungus Cordyceps sinensis. Mycological Research 111: 409–415. Sung GH, Hywel-Jones NL, Sung JM, Luangsa-ard JJ, Shrestha B,

Pradhan BK, 2016. Caterpillar Mushroom, Ophiocordyceps sinensis

Stensrud Ø, Schumacher T, Shalchian-Tabrizi K, Svegården IB,

A Potential

Bioresource

Spatafora JW, 2007. Phylogenetic classification of Cordyceps

for

and the clavicipitaceous fungi. Studies in Mycology 57: 5–59.

Commercialization in Sikkim Himalaya, India. International

Tan N-Z, Berger JL, Zhang Y, Prolla TA, Weindruch R, Zhao C-S,

Journal of Medicinal Mushrooms 18: 337-346.

Bartlett M, Zhu J-S, 2011. The lifespan-prolonging effect of

Pratt AE, 1892. To the snows of Tibet through China: 16–17. Longmans, Green, and Company, London.

Cordyceps sinensis Cs-4 in normal mice and its molecular mechanisms. FASEB Journal 25: 599.1.

Quandt CA, Kepler RM, Gams W, Araújo JPM, Ban S, Evans HC,

Taylor JW, 2011. One fungus = one name: DNA and fungal

Hughes D, Humber R, Hywel-Jones N, Li Z-Z, Luangsa-ard

nomenclature twenty years after PCR. IMA Fungus 2: 113–

JJ, Rehner SA, Sanjuan T, Sato H, Shrestha B, Sung GH, Yao

120.

Y-J, Zare R, Spatafora JW, 2014. Phylogenetic-based nomenclatural

proposals

for

Wei J-C, Wei X-L, Zheng W-F, Guo W, Liu R-D, 2016. Species

Ophiocordycipitaceae

identification and component detection of Ophiocordyceps

(Hypocreales) with new combinations in Tolypocladium. IMA

sinensis cultivated by modern industry. Mycosystema 35: 404–

Fungus 5: 121–134.

410.

Ren Y, Wan D-G, Lu X-M, Guo J-L, 2013. The study of scientific

Wei X-L, Yin X-C, Guo Y-L, Shen N-Y, Wei J-C, 2006. Analyses

name discussion for TCM Cordyceps. Lishizhen Medicine and

of molecular systematics on Cordyceps sinensis and its related

Materia Medica Research 24: 2211–2212.

taxa. Mycosystema 25: 192–202.

Saccardo PA, 1878. Enumeratio pyrenomycetum hypocreaceorum

Winkler D, 2008. The mushrooming fungi market in Tibet

hucusque congitorum systemate carpologico dispositorum.

exemplified by Cordyceps sinensis and Tricholoma matsutake.

Michelia 1: 277–325.

Journal of the International Association of Tibetan Studies 4:

Saccardo PA, 1883. Sylloge fungorum omnium hucusque cognitorum (Vol. 2): 577. Sumptibus Auctoris, Patavii. Saunders

WW,

1842.

Proceedings

of

learned

societies:

1–47. Xia F, Liu Y, Shen GR, Guo L-X, Zhou X-W, 2015. Investigation and analysis of microbiological communities in natural

17

18


Ophiocordyceps sinensis. Canadian Journal of Microbiology

research advances, issues and perspectives. Mycosystema 32:

61: 104–111.

577–597.

Xiang L, Li Y, Zhu Y-J, Luo H-M, Li C-F, Xu X-L, Sun C, Song J-

Zhang W-W, Cheng X-L, Liu X-Z, Xiang M-C, 2016. Genome

Y, Shi L-C, He L, Sun W, Chen S-L, 2014. Transcriptome

studies on nematophagous and entomogenous fungi in China.

analysis of the Ophiocordyceps sinensis fruiting body reveals

Journal of Fungi 2: 1–14.

putative genes involved in fruiting body development and cordycepin biosynthesis. Genomics 103: 154–159.

Zhang Y-J, Li E-W, Wang C-S, Li Y-L, Liu X-Z, 2012. Ophiocordyceps sinensis, The flagship fungus of China:

Xiao W, Yang J-L, Zhu P, Cheng K-D, He H-X, Zhu H-X, Wang

terminology, life strategy and ecology. Mycology 3: 2-10.

Q, 2009. Non-support of species complex hypothesis of

Zhang Y-J, Sun B-D, Zhang S, Wang M, Liu X-Z, Gong W-F, 2010.

Cordyceps sinensis by targeted rDNA-ITS sequence analysis.

Mycobiotal investigation of natural Ophiocordyceps sinensis

Mycosystema 28: 724–730.

based on culture-dependent investigation. Mycosystema 29:

Xizang Institute of Tibetan Hospital and Medicine, 2002. Chinese materia medica (Tibetan Medicine Edition): 147–148. Shanghai Science Technology Press, Shanghai.

518–527. Zhang Y-J, Xu L-L, Zhang S, Liu X-Z, An Z-Q, Wang M, Guo YL, 2009. Genetic diversity of Ophiocordyceps sinensis, a

Yang J-L, Xiao W, He H-X, Zhu H-X, Wang S-F, Cheng K-D, Zhu

medicinal fungus endemic to the Tibetan Plateau: implications

P, 2008. [Molecular phylogenetic analysis of Paecilomyces

for its evolution and conservation. BMC Evolutionary Biology

hepiali and Cordyceps sinensis]. Yao Xue Xue Bao = Acta

9: 290. doi:10.1186/1471-2148-9-290

Pharmaceutica Sinica 43: 421–426. Pubmed:18664207.

Zhao K, Li Y, Zhang H, 2013. Role of dongchongxiacao

Yao Y-S, Zhou Y-J, Gao L, Lu J-H, Wu Z-M, Zhu J-S, 2011.

(Cordyceps) in prevention of contrast-induced nephropathy in

Dynamic alterations of the differential fungal expressions of

patients with stable angina pectoris. Journal of Traditional

Ophiocordyceps sinensis and its mutant genotypes in stroma

Chinese Medicine 33: 283–286.

and caterpillar during maturation of natural Cordyceps sinensis. Journal of Fungal Research 9: 37–49.

Zhou X-W, Li L-J, Tian E-W, 2014. Advances in research of the artificial cultivation of Ophiocordyceps sinensis in China.

Yao Y-S, Zhu J-S, 2016. Indiscriminate use of the Latin name for

Critical Reviews in Biotechnology 34: 233–243.

natural Cordyceps sinensis insect-fungi complex and multiple

Zhu J-S, Gao L, Li X-H, Yao Y-S, Zhou Y-J, Zhao J-Q, Zhou Y-J,

Ophiocordyceps sinensis fungi. China Journal of Chinese

Lu J-H, 2010. Maturational alterations of oppositely orientated

Materia Medica 41: 1361–1366.

rDNA and differential proliferations of CG:AT-biased

Zhang S, Zhang Y-J, 2015. Molecular evolution of three proteincoding

genes

in

the

Chinese

caterpillar

fungus

Ophiocordyceps sinensis. Microbiology, (China) 42: 1549– 1560.

genotypes of Cordyceps sinensis fungi and Paecilomyces hepiali in natural Cordyceps sinensis. American Journal of Biomedical Sciences 2: 217–238. Zhu J-S, Guo Y-L, Yao Y-S, Zhou Y-J, Lu J-H, Qi Y, Chen W, Liu

Zhang S, Zhang Y-J, Liu X-Z, Zhang H, Liu D-S, 2013a. On the

X-J, Wu Z-M, Zhang L, Yin W-T, Zheng T-Y, Zhang L-J,

reliability of DNA sequences of Ophiocordyceps sinensis in

2007. Maturation of Cordyceps sinensis associates with co-

public data bases. Journal of Industrial Microbiology and

existence of Hirsutella sinensis and Paecilomyces hepiali

Biotechnology 40: 365–378.

DNA and dynamic changes in fungal competitive proliferation

Zhang S, Zhang Y-J, Shrestha B, Xu J-P, Wang C-S, Liu X-Z, 2013b. Ophiocordyceps sinensis and Cordyceps militaris:

predominance and chemical profiles. Journal of Fungal Research 5: 214–224.


Zhu J-S, Halpern GM, Jones K, 1998a. The scientific rediscovery

of Cell Biology 37: 284–298.

of an ancient Chinese herbal medicine: Cordyceps sinensis:

Zhu J-S, Yao Y-S, Li Y-L, Wu Z-M, Liu X, Xu H-F, Zhang Z-H,

part I. Journal of Alternative and Complementary Medicine 4:

Ma S-L, 2016. Hereditary variations with DNA segment

289–303.

substitution between fungi in the two types of Cordyceps

Zhu J-S, Halpern GM, Jones K, 1998b. The scientific rediscovery

sinensis ascospores suggest fungal chromosomal fusions

of a precious ancient chinese herbal regimen: cordyceps

Proceedings of 2016 The 11th National Conference of

sinensis Part II. Journal of Alternative and Complementary

Medicinal Mycology. November 29: 92–103. Chengdu,

Medicine 4: 429–457.

Sichuan, China.

Zhu J-S, Li Y-L, 2017. A precious transitional Chinese medicine,

Zhu J-S, Zhao J-G, Gao L, Li X-H, Zhao J-Q, Lu J-H, 2012.

Cordyceps sinensis: Multiple heterogeneous Ophiocordyceps

Dynamically altered expressions of at least 6 Ophiocordyceps

sinensis in the insect-fungi complex. Saarbrüchen, Germany:

sinensis mutants in the stroma of Cordyceps sinensis. Journal

Lambert Academic Publishing.

of Fungal Research 10: 100–112.

Zhu J-S, Wu J-Y, 2015. Genetic heterogeneity of natural Cordyceps sinensis with co-existence of multiple fungi. Chinese Journal

19