The Molecular Heterogeneity of Natural Cordyceps sinensis with Multiple Ophiocordyceps sinensis Fungi Challenges the Anamorph-Teleomorph Connection Hypotheses

Abstract

Natural Cordyceps sinensis is a traditional Chinese medicine with a long history of use as a folk medicine in China. However, whether Ophiocordyceps sinensis is a single fungus or a collective name for multiple fungi remains controversial, as does the anamorph-teleomorph connection of O. sinensis. Although Hirsutella sinensis has been widely considered the sole anamorph of O. sinensis, direct evidence supporting this hypothesis is lacking. This paper reviews the molecular heterogeneity findings of C. sinensis studies and the issues with the hypotheses of the anamorph-teleomorph connection of O. sinensis. Molecular analyses have revealed the coexistence of multiple fungi and multiple genotypes of O. sinensis in natural C. sinensis. Mutant sequences from at least 11 O. sinensis genotypes are registered in GenBank and represent individual fungi distinct from the genome sequence of H. sinensis, which is inconsistent with the "sole anamorph" and "ITS pseudogenes" hypotheses. Together, the multicellular C. sinensis ascospores with mono-/bi-/trinucleate structures in each ascospore and the detection of at least 2 O. sinensis genotypes in the culture of heterokaryotic single-ascospore isolates challenge the study conclusions based on microcycle conidiation of C. sinensis ascospores. During C. sinensis maturation, the fungi that grow differentially in the caterpillar body and stroma of C. sinensis undergo asynchronous, dynamic alterations, supporting the integrated microecosystem hypothesis for natural C. sinensis proposed by Prof. Zongqi Liang.

Suitability and Advantages of Abundance-Weighted Algorithms for the Holistic Comparison of Sample Systems

“…Among these methodologies, RAPD molecular marker polymorphism analysis is the most frequently used technique for comparing overall similarities or dissimilarities (genetic distances) and exploring the phylogenetic (cluster) relationship between the test systems [2,29,[32][33][34][35][36][37][38][39][40][41], although it has been suggested that ISSR may be more sensitive than RAPD [42][43][44] and costly metagenomic approaches may demonstrate advantages in qualitative studies of microbial genetic diversity and molecular ecology [45][46][47]. Among the key factors in study design for molecular marker polymorphism comparisons, the importance of unbiased selection of a plurality of random primers for random amplification of the genomic DNA templates isolated from the examined systems has been addressed [1][2][3][4]40,41]. The use of only a few random primers without reporting the objectivity and representativeness of the selection could lead to bias in the data analysis and thus biased conclusions [1][2][3][4]40,41,48].…”

“…Scientists have reported the molecular heterogeneity of C. sinensis-associated fungi of the genera Hirsutella, Paecilomyces and Tolypocladium as well as Geomyces pannorum, Cladosporium macrocarpum, Phaeosphaeria pontiformis and Neosetophoma samarorum (in total, more than 90 species spanning at least 37 genera) [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. SNP mass spec genotyping and amplicon sequencing with or without molecular cloning have identified the mutant ITS sequences of at least 17 genotypes of O. sinensis fungi with multiple, scattered point mutations or DNA segment substitution hereditary variations from the natural C. sinensis insect-fungi complex [1][2][3][4][10][11][12][13]19,[22][23][24][25][26][27][28]. The successful production of artificial C. sinensis enabled the molecular examination of the fungal strains that were used as inoculation agents and the correlation of the genotype results with the finished product, artificial C. sinensis [28].…”

“…Among the key factors in study design for molecular marker polymorphism comparisons, the importance of unbiased selection of a plurality of random primers for random amplification of the genomic DNA templates isolated from the examined systems has been addressed [1][2][3][4]40,41]. The use of only a few random primers without reporting the objectivity and representativeness of the selection could lead to bias in the data analysis and thus biased conclusions [1][2][3][4]40,41,48]. When C. sinensis insect-fungi complex samples and fungal strain samples were not profiled as a whole, this resulted in the inaccuracy of holistic comparisons, interpretations and conclusions [29].…”

“…Since 1999, PCR-based nrDNA Internal Transcribed Spacer (ITS) sequencing has been used to determine the taxonomic status of the examined fungal specimens in the natural Cordyceps sinensis insect-fungi complex as an auxiliary molecular method to examine the anamorph-teleomorph connection of Ophiocordyceps sinensis [1][2][3][4]. Scientists have reported the molecular heterogeneity of C. sinensis-associated fungi of the genera Hirsutella, Paecilomyces and Tolypocladium as well as Geomyces pannorum, Cladosporium macrocarpum, Phaeosphaeria pontiformis and Neosetophoma samarorum (in total, more than 90 species spanning at least 37 genera) [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21].…”

“…However, most of data generated from holistic studies do not fall into an "all-or-none" qualitative research category; thus, abundance-unweighted algorithms may not serve as suitable analytic tools for these types of research. Using an RAPD molecular polymorphism study as the example, this paper reviews the prerequisites and limitations of using the binary abundance-unweighted algorithms and the advantage of abundance-weighted algorithms in holistic studies.Since 1999, PCR-based nrDNA Internal Transcribed Spacer (ITS) sequencing has been used to determine the taxonomic status of the examined fungal specimens in the natural Cordyceps sinensis insect-fungi complex as an auxiliary molecular method to examine the anamorph-teleomorph connection of Ophiocordyceps sinensis [1][2][3][4]. Scientists have reported the molecular heterogeneity of C. sinensis-associated fungi of the genera Hirsutella, Paecilomyces and Tolypocladium as well as Geomyces pannorum, Cladosporium macrocarpum, Phaeosphaeria pontiformis and Neosetophoma samarorum (in total, more than 90 species spanning at least 37 genera) [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21].…”