Phylogeography and evolution of a fungal–insect association on the Tibetan Plateau

Parasitoidism refers to a major form of interspecies interactions where parasitoids sterilize and/or kill their hosts typically before hosts reach reproductive age. However, relatively little is known about the evolutionary dynamics of parasitoidism. Here, we investigate the spatial patterns of genetic variation of Chinese cordyceps, including both the parasitoidal fungus Ophiocordyceps sinensis and its host insects. We sampled broadly from alpine regions on the Tibetan Plateau and obtained sequences on seven fungal and three insect DNA fragments from each of the 125 samples. Seven and five divergent lineages/cryptic species were identified within the fungus and host insects, respectively. Our analyses suggested that O. sinensis and host insects originated at similar geographic regions in southern Tibet/Yunnan, followed by range expansion to their current distributions. Cophylogenetic analyses revealed a complex evolutionary relationship between O. sinensis and its host insects. Significant congruence was found between host and parasite phylogenies and the time estimates of divergence were similar, raising the possibility of the occurrence of cospeciation events, but the incongruences suggested that host shifts were also prevalent. Interestingly, one fungal genotype was broadly distributed, consistent with recent gene flow. In contrast, the high‐frequency insect genotypes showed limited geographic distributions. The dominant genotypes from both the fungus and the insect hosts may represent ideal materials from which to develop artificial cultivation of this important Chinese traditional medicine. Our results demonstrate that both historical and contemporary events have played important roles in the phylogeography and evolution of the O. sinensis–ghost moth parasitoidism on the Tibetan Plateau.     

冬虫夏草生物学及生态学研究中的关键科学问题研究进展

冬虫夏草是一种具有极高药用和食用价值的珍稀菌类,其生物学特性和可持续发展关键技术一直是冬虫夏草研究关注的重点。总结了冬虫夏草生物学及生态学中的几个重要科学问题,包括冬虫夏草菌侵染蝙蝠蛾幼虫的途径及机制、冬虫夏草菌子实体形成及生长过程中的关键调控因子、冬虫夏草菌遗传多样性及基因组学研究、天然冬虫夏草及其微环境中的微生物群落组成、全球变暖背景下未来青藏高原地区的气候变化对冬虫夏草资源的影响等的研究进展,并分析了现有研究中尚未明确和不足之处。未来在冬虫夏草生物学及生态学的研究中,需要利用和研发更加先进的观测设备及试验方法,以实现天然冬虫夏草发生发育的原位观测和机理研究;充分利用冬虫夏草人工培植技术,结合分子生物学研究,从基因水平上揭示冬虫夏草菌生态适应性及侵染蝙蝠蛾幼虫的机理;建立冬虫夏草科学研究基地,开展长期定位观测试验并结合生态学模型构建明确未来气候变化对冬虫夏草资源的影响,以期进一步深入冬虫夏草生物学和生态学研究、推进我国冬虫夏草资源的合理利用及可持续发展的政策制定。     

On the reliability of fungal materials used in studies on <Emphasis Type="Italic">Ophiocordyceps sinensis</Emphasis>

Ophiocordyceps sinensis (≡Cordyceps sinensis) is one of the best known traditional Chinese medicines, with great benefits to human health and huge economic value. The reliability of fungal materials used in studies of the species is particularly important because contradictory results have been found in various studies in the past decades. Examination of fungal materials specified in reports on O. sinensis showed great variation in both sources and culture conditions of living strains. To test the reliability of the materials used, experiments were carried out to study the effect of culture conditions on the growth of living strains of O. sinensis by using six reliable strains representing the major production regions of the fungus on the Tibetan Plateau. The results showed that O. sinensis is a slow-growing fungus at comparatively low temperature, and that temperature and growth period are crucial factors which can be verified by experiment. Analyses of fungal materials used in 152 papers on O. sinensis from PubMed since 1998 showed that 41 papers lacked detailed information on the fungal materials; 26 used natural products, 11 used artificially cultivated fruit bodies, and 80 used fermentation products from living strains. Of the latter category (using fermentation products), 64 of the papers were found to use unreliable (45) or uncertain (19) strains for fermentation products based on the temperature and growth period for O. sinensis strains verified in this study. Apart from the natural products of O. sinensis, which require scientific identification, a total of at least 116 papers (over three-quarters) used unreliable, uncertain or unspecified materials, including so-called cultivated fruit bodies which were apparently from other species. The reliability of materials or living strains used in studies on O. sinensis is discussed in this paper, and suggestions are made for use of reliable fungal materials in further studies of this fungus.     

西藏高原冬虫夏草资源适宜性区划分析

冬虫夏草是分布在西藏高原高寒草甸中稀缺的可再生生物资源,具有很高的药用价值和经济价值,然而资源的数量、空间分布格局及分布适宜度尚不清晰。根据西藏高原冬虫夏草产区生态环境条件,以海拔高度、植被类型、土壤类型、年平均降水量、年平均气温为区划主要指标,利用GIS空间分析方法对西藏高原冬虫夏草生长区进行适宜性综合区划。结果表明:那曲地区的比如县、索县,昌都市的边坝县、丁青县、类乌齐县、江达县、洛隆县、察雅县、贡觉县部分产区处在适宜区;那曲地区的那曲县、嘉黎县、巴青县、聂荣县,拉萨市的当雄县及其他产区处在次适宜区;日喀则市的大部分地区、林芝市和山南市的低海拔地区为不适宜区。在出产冬虫夏草的地区(市)中,那曲地区、昌都市分布面积占当地产冬虫夏草县域国土面积的52.1%—59.3%,山南市、日喀则市、拉萨市和林芝市分布面积占当地产冬虫夏草县域国土面积的13.7%—26.1%,整个西藏高原产区的冬虫夏草分布面积平均占当地产冬虫夏草县域国土面积的34.5%。综合区划图能比较真实的反映西藏高原冬虫夏草空间分布格局,区划结果与采挖地乡镇调查结果符合程度高,与冬虫夏草单位草原面积统计产量分布较为一致。这一研究方法可为以后冬虫夏草资源调查提供参考,为冬虫夏草资源评估及区划等相关研究提供方法支持。     

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.     

Detection of Ophiocordyceps sinensis in the roots of plants in alpine meadows by nested-touchdown polymerase chain reaction

Ophiocordyceps sinensis, one of the most important income sources of rural Tibetan families, is an entomopathogenic fungus that parasitizes the ghost moth Thitarodes larvae, which live in alpine meadows on the Tibetan Plateau and in the Himalayas. The annual yield of O. sinensis has gradually declined in recent years. However, there is no effective method to sustain or increase the yield of O. sinensis artificially because the life cycle of the O. sinensis anamorph remains unclear. Here we detected O. sinensis in alpine plant roots by nested-touchdown polymerase chain reaction (PCR). Forty-two alpine plant species were screened. The roots from 23 alpine plant species (54.76 %) tested positive including 13 families and 18 genera. The detection results indicate that O. sinensis is present in the plant roots during the anamorph life cycle, to deal with harsh conditions in alpine habitats and have an increased opportunity to infect the larvae. The finding provides new information regarding the biology and ecology of O. sinensis that may be used to sustain this valuable resource.     

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.     

A Polycephalomyces hyperparasite of Ophiocordyceps sinensis leads to shortened duration of production and reduced numbers of host ascospores

Ophiocordyceps sinensis, an entomopathogenic fungus of the Tibetan Plateau is one of the most valuable traditional Chinese medicines. A fungal pathogen of O. sinensis, Polycephalomyces sp. GIMCC 3.570 (isolate 2010A-0818) was characterized by morphology, related to sequences of six genes (SSU, ITS, LSU, TEF1-α, RPB1 and ATP6) found in a BLAST search and five genes (SSU, LSU, TEF1, RPB1 and RPB2) by phylogenetic analysis using maximum parsimony, maximum likelihood and Bayesian methods. The results strongly support placement of P. sp. GIMCC 3.570 in the genus Polycephalomyces. To determine the influence of P. sp. GIMCC 3.570 on the reproduction of its host, 30 artificially infected O. sinensis samples were compared with 30 uninfected controls. In the infected fungus, the life span of the stroma was significantly shorter and significantly fewer ascospores were produced compared with uninfected O. sinensis (p < 0.01).     

Population history and genomic signatures for high-altitude adaptation in Tibetan pigs

Background

The Tibetan pig is one of domestic animals indigenous to the Qinghai-Tibet Plateau. Several geographically isolated pig populations are distributed throughout the Plateau. It remained an open question if these populations have experienced different demographic histories and have evolved independent adaptive loci for the harsh environment of the Plateau. To address these questions, we herein investigated ~ 40,000 genetic variants across the pig genome in a broad panel of 678 individuals from 5 Tibetan geographic populations and 34 lowland breeds.

Results

Using a series of population genetic analyses, we show that Tibetan pig populations have marked genetic differentiations. Tibetan pigs appear to be 3 independent populations corresponding to the Tibetan, Gansu and Sichuan & Yunnan locations. Each population is more genetically similar to its geographic neighbors than to any of the other Tibetan populations. By applying a locus-specific branch length test, we identified both population-specific and -shared candidate genes under selection in Tibetan pigs. These genes, such as PLA2G12A, RGCC, C9ORF3, GRIN2B, GRID1 and EPAS1, are involved in high-altitude physiology including angiogenesis, pulmonary hypertension, oxygen intake, defense response and erythropoiesis. A majority of these genes have not been implicated in previous studies of highlanders and high-altitude animals.

Conclusion

Tibetan pig populations have experienced substantial genetic differentiation. Historically, Tibetan pigs likely had admixture with neighboring lowland breeds. During the long history of colonization in the Plateau, Tibetan pigs have developed a complex biological adaptation mechanism that could be different from that of Tibetans and other animals. Different Tibetan pig populations appear to have both distinct and convergent adaptive loci for the harsh environment of the Plateau.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-834) contains supplementary material, which is available to authorized users.     

Determining novel molecular markers in the Chinese caterpillar fungus Ophiocordyceps sinensis by screening a shotgun genomic library

The Chinese caterpillar fungus Ophiocordyceps sinensis, endemic to alpine regions on the Tibetan Plateau, is one of the most valuable medicinal fungi in the world. Genetic differentiation within this fungus was observed; however, due to lack of highly efficient molecular markers, the overall genetic structure of this fungus has not been clarified. In this study, a shotgun genomic library of O. sinensis was constructed, and >181,848 nt were analyzed from >250 random clones. Primers from 33 sequenced fragments were then designed to amplify O. sinensis samples collected from widely separated regions on the Tibetan Plateau. Ten of the 33 fragments had no amplification or poor sequencing quality from all or certain samples. Sequence variations of the remaining 23 fragments among different samples were investigated in detail. Three fragments (OSRC14, OSRC19, and OSRC32) were the most variable with 7-43 single-nucleotide polymorphism (SNP) sites, representing the SNP frequency of 1.2-6.7?% per nucleotide site. These three fragments have the potential to be useful molecular markers for studying the population genetics of O. sinensis. These results also showed that constructing and screening a shotgun genomic library was an efficient approach to identify novel molecular markers from non-model organisms.     

Freshwater biogeography and limnological evolution of the Tibetan Plateau--insights from a plateau-wide distributed gastropod taxon (Radix spp.)

Background

The Tibetan Plateau is not only the highest and largest plateau on earth; it is also home to numerous freshwater lakes potentially harbouring endemic faunal elements. As it remains largely unknown whether these lakes have continuously existed during the Last Glacial Maximum (LGM), questions arise as to whether taxa have been able to exist on the plateau since before the latest Pleistocene, from where and how often the plateau was colonized, and by which mechanisms organisms conquered remote high altitude lentic freshwater systems. In this study, species of the plateau-wide distributed freshwater gastropod genus Radix are used to answer these biogeographical questions.

Methodology/Principal Findings

Based on a broad spatial sampling of Radix spp. on the Tibetan Plateau, and phylogenetic analyses of mtDNA sequence data, three probably endemic and one widespread major Radix clade could be identified on the plateau. Two of the endemic clades show a remarkably high genetic diversity, indicating a relatively great phylogenetic age. Phylogeographical analyses of individuals belonging to the most widely distributed clade indicate that intra-plateau distribution cannot be explained by drainage-related dispersal alone.

Conclusions/Significance

Our study reveals that Radix spp. persisted throughout the LGM on the Tibetan Plateau. Therefore, we assume the continuous existence of suitable water bodies during that time. The extant Radix diversity on the plateau might have been caused by multiple colonization events combined with a relatively long intra-plateau evolution. At least one colonization event has a Palaearctic origin. In contrast to freshwater fishes, passive dispersal, probably by water birds, might be an important mechanism for conquering remote areas on the plateau. Patterns found in Radix spp. are shared with some terrestrial plateau taxa, indicating that Radix may be a suitable model taxon for inferring general patterns of biotic origin, dispersal and survival on the Tibetan Plateau.     

A real-time qPCR assay to quantify Ophiocordyceps sinensis biomass in Thitarodes larvae

Ophiocordyceps sinensis, an entomogenous fungus parasitic in the larvae of moths (Lepidoptera), is one of the most valuable medicinal fungi, and it only distributed naturally on the Tibetan Plateau. The parasitical amount of O. sinensis in various tissues of the host Thitarodes larvae has an important role in study the occurrence and developmental mechanisms of O. sinensis, but there no an effective method to detect the fungal anamorph. A real-time quantitative PCR (qPCR) system, including a pair of species-specific ITS primers and its related program, was developed for O. sinensis assay with high reliability and efficiency. A calibration curve was established and exhibited a very good linear correlation between the fungal biomass and the C _T values (R ²=0.999419) by the qPCR system. Based on this method, O. sinensis was detected rapidly in four tissues of its host caterpillars, and the results were shown as following: the maximum content of O. sinensis parasitized in the fat-body, and next came body-wall; both of them were much larger than that observed in the haemolymph and intestinal-wall. Taken together, these results show that qPCR assays may become useful tools for study on developmental mechanism of O. sinensis.     

Phylogeography of the Solanaceae-infecting Basidiomycota fungus <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG-3 based on sequence analysis of two nuclear DNA loci

Background  

The soil fungus Rhizoctonia solani anastomosis group 3 (AG-3) is an important pathogen of cultivated plants in the family Solanaceae. Isolates of R. solani AG-3 are taxonomically related based on the composition of cellular fatty acids, phylogenetic analysis of nuclear ribosomal DNA (rDNA) and beta-tubulin gene sequences, and somatic hyphal interactions. Despite the close genetic relationship among isolates of R. solani AG-3, field populations from potato and tobacco exhibit comparative differences in their disease biology, dispersal ecology, host specialization, genetic diversity and population structure. However, little information is available on how field populations of R. solani AG-3 on potato and tobacco are shaped by population genetic processes. In this study, two field populations of R. solani AG-3 from potato in North Carolina (NC) and the Northern USA; and two field populations from tobacco in NC and Southern Brazil were examined using sequence analysis of two cloned regions of nuclear DNA (pP42F and pP89).     

Detection of potentially valuable polymorphisms in four group I intron insertion sites at the 3'-end of the LSU rDNA genes in biocontrol isolates of Metarhizium anisopliae

Background  

The entomopathogenic anamorphic fungus Metarhizum anisopliae is currently used as a biocontrol agent (BCA) of insects. In the present work, we analyzed the sequence data obtained from group I introns in the large subunit (LSU) of rDNA genes with a view to determining the genetic diversity present in an autochthonous collection of twenty-six M. anisopliae isolates selected as BCAs.     

People,money, and protected areas: the collection of the caterpillar mushroom <Emphasis Type="Italic">Ophiocordyceps sinensis</Emphasis> in the <Emphasis Type="Italic">Baima Xueshan</Emphasis> Nature Reserve,Southwest China

The caterpillar mushroom Ophiocordyceps sinensis (syn. Cordyceps sinensis) is among the most valuable mushrooms in the world, and plays a major role for the local economies in its distribution area on the Tibetan Plateau and adjacent regions. Large proportions of its habitat fall into protected areas, and best practice of sustainable harvest is under discussion, considering both, O. sinensis as a valuable income source for rural poor and protection of its populations and habitat. This study aims for a detailed analysis of O. sinensis collection in a nature reserve in Southwest China. We found that harvesting is unevenly distributed among households and villages, with households who have access to the resource but lack adequate alternatives for income generation such as rewarding wage labor, fertile agricultural fields or harvest of other high value products being most involved. Although collection is de jure forbidden, authorities of the nature reserve apply adaptive management strategies for sustainable resource use. This includes the allocation of collection areas to communities based on their traditional land use strategies and the control of harvesters from outside, triggering self-policing of the resource by the local people. The strategies applied provide a promising model also for other protected areas where the caterpillar mushroom is collected.     

Genetic Diversity and Evolution of Chinese Traditional Medicinal Fungus Polyporus umbellatus (Polyporales,Basidiomycota)

Background

Polyporus umbellatus is an important medicinal fungus distributed throughout most area of China. Its wide distribution may have resulted in substantial intraspecific genetic diversity for the fungus, potentially creating variation in its medical value. To date, we know little about the intraspecific genetic diversity of P. umbellatus.

Methodology/Principal Findings

The objective of this research was to assess genetic differences of P. umbellatus from geographically diverse regions of China based on nrDNA ITS and 28S rRNA (LSU, large subunit) sequences. Significant sequence variations in the ITS and LSU sequences were detected. All sclerotial samples were clustered into four clades based on phylogenetic analysis of ITS, LSU and a combined data set of both regions. Heterogeneity of ITS and LSU sequences was detected in 5 and 7 samples respectively. All clone sequences clustered into the same clade except for one LSU clone sequences (from Henan province) which clustered into two clades (Clade I and Clade II). Significant genetic divergence in P. umbellatus was observed and the genetic diversification was greater among sclerotial samples from Shaanxi, Henan and Gansu provinces than among other provinces. Polymorphism of ITS and LSU sequences indicated that in China, P. umbellatus may spread from a center (Shaanxi, Henan and Gansu province) to other regions.

Conclusions/Significance

We found sclerotial samples of P. umbellatus contained levels of intraspecific genetic diversity. These findings suggested that P. umbellatus populations in Shaanxi, Henan and Gansu are important resources of genetic diversity and should be conserved accordingly.     

Phylogenetic and biogeographic implications inferred by mitochondrial intergenic region analyses and ITS1-5.8S-ITS2 of the entomopathogenic fungi <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">B. brongniartii</Emphasis>

Background  

The entomopathogenic fungi of the genus Beauveria are cosmopolitan with a variety of different insect hosts. The two most important species, B. bassiana and B. brongniartii, have already been used as biological control agents of pests in agriculture and as models for the study of insect host - pathogen interactions. Mitochondrial (mt) genomes, due to their properties to evolve faster than the nuclear DNA, to contain introns and mobile elements and to exhibit extended polymorphisms, are ideal tools to examine genetic diversity within fungal populations and genetically identify a species or a particular isolate. Moreover, mt intergenic region can provide valuable phylogenetic information to study the biogeography of the fungus.     

Isolation of the MAT1-1 mating type idiomorph and evidence for selfing in the Chinese medicinal fungus Ophiocordyceps sinensis

Ophiocordyceps sinensis is one of the most valued medicinal fungi in China. Research on the mating system and sexual development is vitally important to this endangered species. Previous efforts devoted to investigate the mating type (MAT) locus of O. sinensis, however, resulted in an incomplete understanding. In this study, the MAT1-1 locus of O. sinensis was investigated. The conserved α-box and HMG-box regions of the MAT1-1-1 and MAT1-1-3 genes, respectively, and a conserved region of the DNA lyase gene were successfully amplified using degenerate PCR. A combination of TAIL-PCR and long-range PCR were used to connect these genes and obtain the sequence of the MAT1-1 locus. Screening of 22 single spore isolates by PCR demonstrated that both the MAT1-1-1 and MAT1-2-1 genes cooccurred within the same isolate. Additionally, both MAT1-1-1 and MAT1-2-1 are expressed in vegetative mycelia, providing evidence that O. sinensis is likely capable of selfing. DAPI (4,6-diamidino-2-phenylindole) staining of ascospores and hyphae showed that a majority of hyphal compartments are binucleate, suggesting that O. sinensis may be pseudohomothallic. Analyses of sequence diversity showed lower levels of genetic diversity in MAT1-1-1 compared to MAT1-2-1, indicating the possibility that different selective pressures act on the two MAT idiomorphs. The MAT1-1-1 sequences of O. sinensis and Tolypocladium inflatum cluster as a monophyletic group consistent with phylogenetic classification of Ophiocordycipitaceae. Comparison of the structure of the MAT1-1 locus across hypocrealean taxa showed that O. sinensis contains all three mating type genes (MAT1-1-1, MAT1-1-2, and MAT1-1-3) and supported previous observations that of the four families in Hypocreales, MAT1-1-3 has undergone a lineage specific loss only in some members of the Cordycipitaceae.