Molecular phylogenies reveal host-specific divergence of Ophiocordyceps unilateralis sensu lato following its host ants

Ophiocordyceps unilateralis (Hypocreales, Ascomycetes) is an entomopathogenic fungus specific to formicine ants (Formicinae, Hymenoptera). Previous works have shown that the carpenter ant Camponotus leonardi acts as the principal host with occasional infections of ants from the genus Polyrhachis (sister genus of Camponotus). Observations were made on the permanent plots of Mo Singto, Khao Yai National Park of Thailand according to which O. unilateralis was found to occur predominantly on three host species: C. leonardi, C. saundersi and P. furcata. Molecular phylogenies of the elongation factor 1-α and β-Tubulin genes indicate a separation of O. unilateralis samples into three clades, reflecting specificity to each of the three different ant species. Samples collected from P. furcata and from C. leonardi were found to form sister groups with samples from C. saundersi forming an outgroup to the latter. Additional samples collected from unidentified ant species of Camponotus and Polyrhachis were positioned as outgroups to those samples on identified species. These results demonstrate that O. unilateralis is clearly not a single phylogenetic species and comprises at least three species that are specific to different host ant species. These cryptic species may arise through recent events of speciation driven by their specificity to host ant species.     

Ophiocordyceps barnesii and its relationship to other melolonthid pathogens with dark stromata

A hypocrealean Coleoptera pathogen with characteristic part-spores, collected from Khao Yai National Park and Kaeng Krachan National Park in Thailand, is reported. The overall morphology was similar to Cordyceps barnesii, which is known from Sri Lanka, with ascospores disarticulating into four unusually long part-spores that were 30-40 μm long. This disarticulation and part-spore size is, so far, unique within Cordyceps sensu lato. The Thai material was identified with C. barnesii and its placement in the genus Ophiocordyceps was confirmed. Multigene analyses based on the ribosomal small subunit, RPB1 and RPB2 genes revealed the close relationship of the Thai material to Ophiocordyceps konnoana as well as O. ravenelii, O. superficialis, and O. nigrella (all of which have significantly smaller part-spores). However, Ophiocordyceps barnesii and these related species were all characterised by dark-brown to purplish stromata and an affinity for melolonthid larval hosts. No anamorph was seen in the field and was not produced in the slow-growing cultures.     

冬虫夏草和中国被毛孢形态学再研究

产自甘肃省冬虫夏草主产区的冬虫夏草具有丰富的形态多样性,少教具有2-4个子座,个别子座叉状分枝.通过移栽培养,对冬虫夏草Ophiocordyceps sinensis子座的发育过程进行了连续观察,并对子囊孢子的萌发及中国被毛孢Hirsutella sinensis的形态特征进行了再研究.观察到:在子囊孢子弹射期,子座可...     

Systematic analyses of Ophiocordyceps lanpingensis sp. nov., a new species of Ophiocordyceps in China

A new species of Ophiocordyceps, Ophiocordyceps lanpingensis collected from Lanping County, Yunnan Province, southwestern China, was described based on morphological characteristic, ITS1-5.8S-ITS2 rDNA sequences analyses, 5-gene (nrSSU, nrLSU, tef-1α, rpb1 and rpb2) sequences analyses and MAT1-2-1 gene sequences analyses. This species was characterized by thinner stroma, smaller perithecium, thinner ascospore (multiseptate with short septation). The phylogenetic analyses based on the ITS, the 5-gene and MAT1-2-1 gene dataset showed O. lanpingensis had the closest evolution relationship with O. robertsii and O. sinensis, but still had obvious distances to them. Both morphological character and systematic analyses supported that O. lanpingensis was a new species of Ophiocordyceps.     

丝虫草及其无性型关系的分子确证

目的对丝虫草（Ophiocordyceps filiform is）及从子囊孢子分离所得的无性型进行测序和比较分析,确证二者之间的对应关系。方法采用PCR技术,以rDNA-ITS区为分子指标。结果系统进化树显示丝虫草的无性型归为被毛孢属较为合理,同时通过形态特征比较的结果表明,其无性型为荔波被毛孢（Hirsutella liboensis）。结论本研究首次确立了丝虫草的有性型与无性型的对应关系,并且成功培育出了与天然丝虫草相同的虫草子实体。     

<Emphasis Type="Italic">Ophiocordyceps pulvinata</Emphasis> sp. nov., a pathogen of ants with a reduced stroma

Ophiocordyceps pulvinata, a pathogen of ants, is formally described as a new species. Genus level designation of this species is difficult due to several apparently conflicting morphological and ecological characters. Affinity with Ophiocordyceps is suggested by the dark color stroma and ascospore morphology. However, the species was included in a book of entomopathogenic fungi of Japan as Torrubiella sp. due to the production of perithecia on an astipitate stroma. Phylogenetic analyses of molecular data support a close relationship with O. unilateralis, a finding consistent with morphological characteristics of the color, asci and ascospores and ecological traits of host affiliation. Thus, O. pulvinata represents another example of the loss of stipe for the hypocrealean arthropod pathogenic fungi and highlights the utility of asci and ascospore morphology as taxonomically informative characters of closely related taxa.     

分离自冬虫夏草可培养真菌的多样性研究

冬虫夏草是生长于青藏高原的一种名贵中药材。天然冬虫夏草及其微环境中生活着多种真菌。作者使用常规分离培养方法对冬虫夏草的真菌区系进行研究。从天然冬虫夏草的子座、菌核和菌膜3个部位共分离到572个真菌菌株,并根据形态特征将大部分菌株鉴定到37个不同的属。这些菌株经SSCP(single-strand conformation polymorphism)分析后,再根据nrDNAITS序列的相似性(以97%为阈值)共区分出92种不同的分类单元(operational taxonomic unit,OTU)。其中,属于子囊菌的菌株数及OTU数均比接合菌和担子菌多。从菌膜分离的菌株数及OTU数都明显多于子座和菌核。分离自子座的优势真菌是产黄青霉Penicillium chrysogenum,而分离自菌核和菌膜的优势真菌均为玫红假裸囊菌Pseudogymnoascus roseus。尚未最终鉴定的部分真菌可能为新的真菌物种。     

Ophiocordyceps sessilis sp. nov., a new species of Ophiocordyceps on Camponotus ants in Japan

Ophiocordyceps sessilis, a new species of Ophiocordycipitaceae, Hypocreales, was found on Camponotus obscuripes ants, where the ants were primarily infected by another close relative, O. pulvinata. Morphological observation clearly indicated that O. sessilis and O. pulvinata are distinct species. Ophiocordyceps sessilis exhibited superficial development of perithecia and ascospore disarticulation into part-spores, not known in O. pulvinata. Nucleotide sequence data suggested that O. sessilis belongs in Ophiocordyceps, Ophiocordycipitaceae. Molecular data also indicated that O. sessilis is a close relative of O. cuboidea, O. prolifica, O. paracuboidea, and O. ryogamiensis, which are all known to produce part-spores. Since O. sessilis is always associated with ants infected by O. pulvinata, O. sessilis may be a hyperparasite of O. pulvinata.     

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.     

Host insect species of Ophiocordyceps sinensis: a review

Ophiocordyceps sinensis (≡ Cordyceps sinensis) is one of the most valued medicinal fungi in China, used for its invigorating effects in strengthening the body and restoring energy. The fungus parasitizes larvae of moths and converts them into sclerotia from which the fungus fruiting body grows. Since the late 1950s, considerable effort has been devoted to the study of host insects related to the fungus. In the present paper, the research history of insect species associated with Ophiocordyceps sinensis is briefly reviewed and an extensive literature survey is presented. Ninety-one insect names, spanning 13 genera, related to host insects of Ophiocordyceps sinensis are investigated. The relationships between the reported insect species and Ophiocordyceps sinensis are analyzed. Fifty-seven of these are considered as recognizable potential host species of the fungus distributed throughout the Tibetan Plateau, whilst eight are considered as indeterminate hosts and 26 as non-hosts. Among the names of recognizable potential host insects, three are invalid (nomen nudum) and require further study. This work provides basic information for management of the insect resources and for the conservation and sustainable use of Ophiocordyceps sinensis.     

冬虫夏草Ophiocordyceps sinensis研究中几个值得关注的问题

扼要概述和讨论了冬虫夏草与寄主的关系,种的遗传变异、隐蔽种及伴生菌复合体,高活性产物的体细胞杂交方法及固体人工培养和产品标准等方面的一些新的研究动态。     

下垂虫草的重寄生现象及重寄生菌的鉴定

在安徽省岳西县鹞落坪自然保护区,发现了1种寄生于下垂虫草(Ophiocordyceps nutans)的重寄生菌。通过形态特征观察和ITS序列分子生物学分析,将其鉴定为枝多头霉(Polycephalomyces ramosus)。其孢梗束着生于下垂虫草子实体的可孕部,顶部膨大呈球形。本种具有产生单个卵形的A型分生孢子的顶侧生排列的A型瓶梗和能形成分生孢子链的B型瓶梗,并在培养基上的气生菌丝和孢梗束的柄部都观察到梭形、链生的B型分生孢子。GenBank中相关种的ITS序列与拉丁名可能有误。     

Pseudotripoconidium, a new anamorph genus connected to Orbilia

A new anamorphic fungus is described based on four isolates from ascospores of Orbilia aff. luteorubella. This fungus differs from previously known Orbilia anamorphs in producing inversely pyramidal, unicellular conidia with several protuberances at their distal end. Conidia produce 1-7 prominent denticles that emerge from a node at the conidiophore apex. Conidiogenesis is holoblastic. Because phylogenetic analysis indicated greater than 90% ITS sequence similarities among the four isolates they are treated here as a single species. In the sequence analysis of the internal transcribed spacer region (ITS) these isolates and other sequences identified as O. aff. luteorubella were nested within Orbilia and formed a clade with 99% bootstrap support. This clade is separated from nematode-trapping species of Orbilia. Based on both morphological and molecular analyses, we propose a new genus, Pseudotripoconidium.     

青海省冬虫夏草资源特征分析

冬虫夏草作为青藏高原特有的生物资源,对生长环境有着极其特殊的要求.通过调查青海省冬虫夏草资源分布状况,选取玉树、果洛、黄南、海南等地区的冬虫夏草进行氨基酸、微量元素含量检测,以反映冬虫夏草资源特征.研究结果表明,青海省冬虫夏草含有丰富的氨基酸和微量元素,且冬虫夏草不同部位的氨基酸、微量元素含量有明显差别.将青海省冬虫夏草与其他产区的冬虫夏草氨基酸含量进行对比分析,青海省冬虫夏草氨基酸含量低于四川、云南产区的冬虫夏草,和西藏产区的冬虫夏草氨基酸含量基本一致.     

High diversity of the fungal community structure in naturally-occurring Ophiocordyceps sinensis

Background

Ophiocordyceps sinensis (syn. Cordyceps sinensis), which is a parasite of caterpillars and is endemic to alpine regions on the Tibetan Plateau, is one of the most valuable medicinal fungi in the world. “Natural O. sinensis specimens” harbor various other fungi. Several of these other fungi that have been isolated from natural O. sinensis specimens have similar chemical components and/or pharmaceutical effects as O. sinensis. Nevertheless, the mycobiota of natural O. sinensis specimens has not been investigated in detail.

Methodology/Principal Findings

Based on the technique of PCR-single-strand conformation polymorphism (PCR-SSCP), the mycobiota of three different sections (stromata, sclerotia, and mycelial cortices) from natural O. sinensis specimens were investigated using both culture-dependent and -independent methods. For the culture-dependent method, 572 fungal strains were isolated, and 92 putative operational taxonomic units (OTUs) were identified from 226 sequenced strains with the threshold of 97%. For the culture-independent method, 490 fungal clones were identified from about 3000 clones of ITS fragments from the whole-community DNA; based on PCR-SSCP analyses, 266 of these clones were selected to be sequenced, and 118 putative OTUs were detected. The overwhelming majority of isolates/clones and OTUs were detected from mycelial cortices; only a few were detected from stromata and sclerotia. The most common OTUs detected with both methods belonged to Ascomycota; however, only 13 OTUs were detected simultaneously by both methods. Potential novel lineages were detected by each of the two methods.

Conclusions/Significance

A great number of fungal species present in the mycobiota of naturally-occurring O. sinensis specimens were detected, and many of them may represent undescribed lineages. That only a few of the same OTUs were detected by both methods indicated that different methods should be used. This study increased our understanding about the fungal community structure of this valuable medicinal herb.     

Relationship between intraspecific variations and host insects of Ophiocordyceps nutans collected in Japan

To investigate the host specificity of Ophiocordyceps nutans against hemipteran insects in the wild, we determined the relationship between host species and rDNA-internal transcribed spacer (ITS) variation in O. nutans. The analyzed fungal specimens infected 16 host species belonging to four families of Hemiptera. The molecular phylogenetic analysis revealed that O. nutans can be classified into two types corresponding to their host families. The genetic distance values between the two types were very remote (>0.084), and the strains of O. nutans that parasitized Halyomorpha halys and Plautia crossota stali, well-known insect pests, formed a subclade. The results suggest that O. nutans should have host specificity which can be valuable for developing biological control agents against specific hemipteran insects.     

On the reliability of DNA sequences of Ophiocordyceps sinensis in public databases

Some DNA sequences in the International Nucleotide Sequence Databases (INSD) are erroneously annotated, which has lead to misleading conclusions in publications. Ophiocordyceps sinensis (syn. Cordyceps sinensis) is a fungus endemic to the Tibetan Plateau, and more than 100 populations covering almost its distribution area have been examined by us over recent years. In this study, using the data from authentic materials, we have evaluated the reliability of nucleotide sequences annotated as O. sinensis in the INSD. As of October 15, 2012, the INSD contained 874 records annotated as O. sinensis, including 555 records representing nuclear ribosomal DNA (63.5 %), 197 representing protein-coding genes (22.5 %), 92 representing random markers with unknown functions (10.5 %), and 30 representing microsatellite loci (3.5 %). Our analysis indicated that 39 of the 397 internal transcribed spacer entries, 27 of the 105 small subunit entries, and five of the 53 large subunit entries were incorrectly annotated as belonging to O. sinensis. For protein-coding sequences, all records of serine protease genes, the mating-type gene MAT1-2-1, the DNA lyase gene, the two largest subunits of RNA polymerase II, and elongation factor-1α gene were correct, while 14 of the 73 β-tubulin entries were indeterminate. Genetic diversity analyses using those sequences correctly identified as O. sinensis revealed significant genetic differentiation in the fungus although the extent of genetic differentiation varied with the gene. The relationship between O. sinensis and some other related fungal taxa is also discussed.