Yi-Jian Yao

Phylogenetic-based nomenclatural proposals for Ophiocordycipitaceae (Hypocreales) with new combinations in Tolypocladium.

Ophiocordycipitaceae is a diverse family comprising ecologically, economically, medicinally, and culturally important fungi. The family was recognized due to the polyphyly of the genus Cordyceps and the broad diversity of the mostly arthropod-pathogenic lineages of Hypocreales. The other two cordyceps-like families, Cordycipitaceae and Clavicipitaceae, will be revised taxonomically elsewhere. Historically, many species were placed in Cordyceps, but other genera have been described in this family as well, including several based on anamorphic features. Currently there are 24 generic names in use across both asexual and sexual life stages for species of Ophiocordycipitaceae. To reflect changes in Art. 59 in the International Code of Nomenclature for algae, fungi, and plants (ICN), we propose to protect and to suppress names within Ophiocordycipitaceae, and to present taxonomic revisions in the genus Tolypocladium, based on rigorous and extensively sampled molecular phylogenetic analyses. When approaching this task, we considered the principles of priority, monophyly, minimizing taxonomic revisions, and the practical utility of these fungi within the wider biological research community.

A survey of the geographic distribution of Ophiocordyceps sinensis

Ophiocordyceps sinensis is one of the best known fungi in Traditional Chinese Medicine. Many efforts have been devoted to locating the production areas of this species resulting in various reports; however, its geographic distribution remains incompletely understood. Distribution of O. sinensis at the county level is clarified in this work based on both a literature search and fieldwork. More than 3600 publications related to O. sinensis were investigated, including scientific papers, books, and online information. Herbarium specimens of O. sinensis and field collections made by this research group during the years 2000–2010 were examined to verify the distribution sites. A total of 203 localities for O. sinensis have been found, of which 106 are considered as confirmed distribution sites, 65 as possible distribution sites, 29 as excluded distribution sites and three as suspicious distribution sites. The results show that O. sinensis is confined to the Tibetan Plateau and its surrounding regions, including Tibet, Gansu, Qinghai, Sichuan, and Yunnan provinces in China and in certain areas of the southern flank of the Himalayas, in the countries of Bhutan, India and Nepal, with 3,000 m as the lowest altitude for the distribution. The fungus is distributed from the southernmost site in Yulong Naxi Autonomous County in northwestern Yunnan Province to the northernmost site in the Qilian Mountains in Qilian County, Qinghai Province, and from the east edge of the Tibetan Plateau in Wudu County, Gansu Province to the westernmost site in Uttarakhand, India. The clarification of the geographic distribution of O. sinensis will lay the foundation for conservation and sustainable use of the species.

Non-concerted ITS evolution in fungi, as revealed from the important medicinal fungus Ophiocordyceps sinensis

The internal transcribed spacer (ITS) of nuclear ribosomal DNA (nrDNA) has been widely used as a molecular marker in phylogenetic studies and has been selected as a DNA barcode for fungi. It is generally believed that nrDNA conforms to concerted evolution in most eukaryotes; however, intraindividual-intraspecific polymorphisms of this region were reported in various organisms, suggesting a non-concerted evolutionary process. In Ophiocordyceps sinensis, one of the most valuable medicinal fungi, a remarkable variation of the ITS region has been revealed. Some highly divergent sequences were thought to represent cryptic species, different species or genotypes in previous studies. To clarify the unusual ITS polymorphisms observed in O. sinensis, specific primers were designed to amplify ITS paralogs from pure cultures of both single-ascospore and tissue isolates in this study. All of the available ITS sequences, including those generated by this group and those in GenBank, were analyzed. Several AT-biased ITS paralogs were classified as pseudogenes based on their nucleotide compositions, secondary structures and minimum free energies of their 5.8S rRNAs, substitution rates, phylogenetic positions and gene expression analyses. Furthermore, ITS pseudogenes were amplified with specific primers from 10 of the 28 strains tested, including eight single-ascospore and two tissue isolates. Divergent ITS paralogs were proved to coexist in individual genomes, suggesting a non-concerted mechanism of evolution in the ITS region of O. sinensis. The hypotheses that divergent ITS paralogs represent cryptic or other species or different genotypes were thus rejected.

Complete mitochondrial genome of the medicinal fungus Ophiocordyceps sinensis.

As part of a genome sequencing project for Ophiocordyceps sinensis, strain 1229, a complete mitochondrial (mt) genome was assembled as a single circular dsDNA of 157,510 bp, one of the largest reported for fungi. Conserved genes including the large and small rRNA subunits, 27 tRNA and 15 protein-coding genes, were identified. In addition, 58 non-conserved open reading frames (ncORFs) in the intergenic and intronic regions were also identified. Transcription analyses using RNA-Seq validated the expression of most conserved genes and ncORFs. Fifty-two introns (groups I and II) were found within conserved genes, accounting for 68.5% of the genome. Thirty-two homing endonucleases (HEs) with motif patterns LAGLIDADG (21) and GIY-YIG (11) were identified in group I introns. The ncORFs found in group II introns mostly encoded reverse transcriptases (RTs). As in other hypocrealean fungi, gene contents and order were found to be conserved in the mt genome of O. sinensis, but the genome size was enlarged by longer intergenic regions and numerous introns. Intergenic and intronic regions were composed of abundant repetitive sequences usually associated with mobile elements. It is likely that intronic ncORFs, which encode RTs and HEs, may have contributed to the enlarged mt genome of O. sinensis.

Development of conventional and nested PCR assays for the detection of Ophiocordyceps sinensis

Ophiocordyceps sinensis, endemic to the Tibetan Plateau, is one of the most important medicinal fungi with a huge economic value. In the present study, specific primer pairs were designed based on a comprehensive ITS sequence dataset of O. sinensis and its related fungi, and tested for specificity and sensitivity through PCR experiments using 27 individuals of O. sinensis from different geographical origins and 40 other related fungal species in terms of phylogeny or ecology. A primer pair highly specific to O. sinensis was obtained, yielding a 275 bp PCR product from all the individuals of O. sinensis but no product from the other fungi tested. The detection limit of the primers was demonstrated to be 10 ng of pure O. sinensis DNA for conventional PCR and 10 pg for nested PCR in a 25 µl reaction system. Soil samples collected from the habitat of O. sinensis were also tested using this PCR assay. The results showed that the primer pair and PCR‐based assays developed in this study can be applied to the rapid detection of O. sinensis in its natural habitat.

Development of microsatellite markers for Ophiocordyceps sinensis (Ophiocordycipitaceae) Using an ISSR-TAIL-PCR method

PREMISE OF THE STUDY Microsatellite primers were developed for Ophiocordyceps sinensis, an endangered medicinal fungus endemic to the Tibetan Plateau, to investigate its genetic diversity and population structure. METHODS AND RESULTS An inter-simple sequence repeat (ISSR)-thermal asymmetric interlaced (TAIL)-PCR method was established to develop microsatellite markers. A total of 30 perfect and imperfect microsatellites were identified in 48 individuals of O. sinensis from five provinces within China representing different populations. Seventeen loci were polymorphic with two to four alleles per locus, while 13 were monomorphic. CONCLUSIONS The results indicate that the microsatellite markers developed here may be used in studies of population genetics and conservation biology of O. sinensis. Furthermore, the ISSR-TAIL-PCR method is a simple strategy for microsatellite marker development.

Comparison of different sequencing and assembly strategies for a repeat-rich fungal genome, Ophiocordyceps sinensis

Ophiocordyceps sinensis is one of the most expensive medicinal fungi world-wide, and has been used as a traditional Chinese medicine for centuries. In a recent report, the genome of this fungus was found to be expanded by extensive repetitive elements after assembly of Roche 454 (223Mb) and Illumina HiSeq (10.6Gb) sequencing data, producing a genome of 87.7Mb with an N50 scaffold length of 12kb and 6972 predicted genes. To test whether the assembly could be improved by deeper sequencing and to assess the amount of data needed for optimal assembly, genomic sequencing was run several times on genomic DNA extractions of a single ascospore isolate (strain 1229) on an Illumina HiSeq platform (25Gb total data). Assemblies were produced using different data types (raw vs. trimmed) and data amounts, and using three freely available assembly programs (ABySS, SOAP and Velvet). In nearly all cases, trimming the data for low quality base calls did not provide assemblies with higher N50 values compared to the non-trimmed data, and increasing the amount of input data (i.e. sequence reads) did not always lead to higher N50 values. Depending on the assembly program and data type, the maximal N50 was reached with between 50% to 90% of the total read data, equivalent to 100× to 200× coverage. The draft genome assembly was improved over the previously published version resulting in a 114Mb assembly, scaffold N50 of 70kb and 9610 predicted genes. Among the predicted genes, 9213 were validated by RNA-Seq analysis in this study, of which 8896 were found to be singletons. Evidence from genome and transcriptome analyses indicated that species assemblies could be improved with defined input material (e.g. haploid mono-ascospore isolate) without the requirement of multiple sequencing technologies, multiple library sizes or data trimming for low quality base calls, and with genome coverages between 100× and 200×.

Molecular and morphological studies of Paecilomyces sinensis reveal a new clade in clavicipitaceous fungi and its new systematic position

The type materials of Paecilomyces sinensis, including herbarium specimen and ex-type strain, were re-examined to clarify its relationships with other species. Morphological observations on the strain grown in various culture media revealed that the fungus was morphologically related to Polycephalomyces, since it produced conidial mass and lanceolate or narrowly lageniform phialides. Six genes, including ITS, nrSSU, nrLSU, tef1, rpb1 and rpb2, were amplified from the type materials and used in phylogenetic analyses to determine the systematic position of the fungus in the framework of clavicipitaceous fungi. The results place P. sinensis with Polycephalomyces formosus, the type species of Polycephalomyces, and Cordyceps ramosopulvinata forming a clade unaffiliated with the known families of clavicipitaceous fungi. Based on both morphological and molecular data, a new combination, Polycephalomyces sinensis, is proposed for Paecilomyces sinensis. The new clade found in this study is designated as Polycephalomyces clade and expands the phylogenetic diversity for clavicipitaceous fungi. The teleomorph–anamorph connection between Berkelella and Polycephalomyces previously conceived cannot be retained as the type species of Polycephalomyces, P. formosus, is closely linked to species of Cordyceps s.l. in the new clade.

Bacterial diversity in native habitats of the medicinal fungus Ophiocordyceps sinensis on Tibetan Plateau as determined using Illumina sequencing data

Ophiocordyceps sinensis is one of the most well-known traditional Chinese medicinal fungi. In this study, bacterial diversity in the soils of native habitats of O. sinensis was investigated using Illumina sequencing data. A total of 525,000 sequences of V6-16S rRNA were analyzed. The number of OTUs from each sample ranged from 13,858 to 15,978 at 97% sequence similarity cut-off. The results demonstrated that the deep sequencing approach provides improved access to rare genotypes. Richness indices and Shannons diversity index did not differ significantly between samples collected from locations where O. sinensis was present (Os1-3) and not present (NOs1-3). Classified bacterial sequences were grouped into 23 phyla including Proteobacteria, Actinobacteria, Acidobacteria, Verrucomicrobia, etc. The Venn diagram revealed that 7183 OTUs belonging to 14 phyla were shared by Os, NOs and MP (mycelial pellicle wrapping the sclerotium of O. sinensis) samples, possibly representing a core microbiome existing in native habitats of O. sinensis, and that 863 belonging to 12 phyla were shared by Os and MP samples, possibly related to the occurrence of O. sinensis. Overall, the results revealed a high bacterial diversity in the soil samples and the relationships between the bacterial diversity and O. sinensis merit further investigation.

rRNA Pseudogenes in Filamentous Ascomycetes as Revealed by Genome Data

The nuclear ribosomal DNA (rDNA) is considered as a paradigm of concerted evolution. Components of the rDNA tandem repeats (45S) are widely used in phylogenetic studies of different organisms and the internal transcribed spacer (ITS) region was recently selected as a fungal DNA bar code. However, rRNA pseudogenes, as one kind of escape from concerted evolution, were reported in a wide range of organisms, especially in plants and animals. Moreover, large numbers of 5S rRNA pseudogenes were identified in several filamentous ascomycetes. To study whether rDNA evolves in a strict concerted manner and test whether rRNA pseudogenes exist in more species of ascomycetes, intragenomic rDNA polymorphisms were analyzed using whole genome sequences. Divergent rDNA paralogs were found to coexist within a single genome in seven filamentous ascomycetes examined. A great number of paralogs were identified as pseudogenes according to the mutation and secondary structure analyses. Phylogenetic analyses of the three rRNA coding regions of the 45S rDNA repeats, i.e., 18S, 5.8S, and 28S, revealed an interspecies clustering pattern of those different rDNA paralogs. The identified rRNA pseudogenic sequences were validated using specific primers designed. Mutation analyses revealed that the repeat-induced point (RIP) mutation was probably responsible for the formation of those rRNA pseudogenes.