真菌DNA条形码技术研究进展

DNA条形码(DNA barcoding)技术作为一门新兴的物种鉴定方法以其灵敏、精确、方便和客观的优势,在动植物和微生物的分类鉴定中已经得到广泛应用.真菌鉴定中常用作标准条形码的是核核糖体DNA内转录间隔区(Internal transcribed spacer,ITS),如今也有一些新型条形码被发现和应用到实际操作中,如微条形码、ND6、EF3.本文对DNA条形码技术的产生和发展做出了总结,通过研究其在真菌中应用的实际案例分析了DNA条形码技术的优缺点及发展趋势,并指出DNA条形码技术将以全新的视角来弥补传统分类学的不足,最终实现生物自身的序列变异信息与现有形态分类学的结合.     

DNA barcoding to map the microbial communities: current advances and future directions

During the last two decades, the DNA barcode development towards microbial community has increased dramatically. DNA barcode development is related to error-free and quick species identification which aid in understanding the microbial biodiversity, as well as the diseases related to microbial species. Here, we seek to evaluate the so-called barcoding initiatives for the microbial communities and the emerging trends in this field. In this paper, we describe the development of DNA marker-based DNA barcoding system, comparison between routine species identification and DNA barcode, and microbial biodiversity and DNA barcode for microbial communities. Two major topics, such as the molecular diversity of viruses and barcode for viruses have been discussed at the same time. We demonstrate the current status and the maker of DNA barcode for bacteria, algae, fungi, and protozoa. Furthermore, we argue about the promises, limitations, and present and future challenges of microbial barcode development.     

Intragenomic variation in nuclear ribosomal markers and its implication in species delimitation,identification and barcoding in fungi

Intragenomic variation is the molecular variation within the genome among repetitive DNA. As a multigene family, nuclear ribosomal DNA (rDNA) has been widely used in fungal taxonomy for their ease in amplification and suitable variability to attain various levels of taxonomic resolution. At the intraspecific level, rDNA is believed to be under concerted evolution and the internal transcribed spacers (ITS) region is actually accepted as a universal barcoding marker for fungi. However, documentation of intragenomic variation of rDNA indicated that it can be problematic in species delimitation and identification. Fungal taxonomic studies have not generally taken into account the intragenomic variation of rDNA in a systematic manner. In this review, our objective is to address the definition, the origin and the mechanisms for maintenance of intragenomic variation, as well as its implication in the domain of fungal molecular taxonomy, particularly for species delimitation, identification and DNA barcoding. With advanced sequencing technologies (second and third generations), we also addressed how these technologies can be used to study the intragenomic variation of rDNA and also how the intragenomic variation will impact on DNA barcoding via high-throughput sequencing.     

DNA条形码分析方法研究进展

DNA条形码是一段短的、标准化的DNA序列,DNA条形码技术通过对DNA条形码序列分析实现物种的有效鉴定.随着生物DNA条形码序列的大量测定,DNA条形码分析方法得到迅速发展,推动了其在生物分子鉴定中的应用.2003年以来,DNA条形码技术已广泛应用于动物、植物和真菌等物种的鉴定,并有力地推动了生物分类学、生物多样性和生态学等学科的发展.本文在综述DNA条形码技术的基础上,总结了5类主要的DNA条形码分析方法,即基于遗传距离的分析、基于遗传相似度的分析、基于系统发育树的分析、基于序列特征的分析和基于统计分类法的分析,并进一步展望了DNA条形码技术的发展与应用.     

The Soil FungiLog procedure: method and analytical approaches toward understanding fungal functional diversity

Conservation methods often are focused on preserving the biodiversity of a particular landscape or ecosystem. Scientists frequently employ species richness as an indicator of biodiversity. However, species richness data are problematic when attempts are made to enumerate microfungi, particularly those from the soil. Many soil fungi fail to sporulate, making identification difficult. Other means of assessing the importance of fungi to ecosystem preservation must be developed. Otherwise, microfungi might be overlooked in discussions of ecosystem management and conservation issues. Herein, we have described a procedure (Soil FungiLog) and analytical techniques that will let investigators examine the functional role that soil fungi play in providing structure and stability to ecosystems. Ecosystem function in many cases might be more important than species diversity in gaining an understanding of ecosystem dynamics. Functional attributes are critical for maintaining ecosystem structure and stability. The preservation of the functions associated with the extant biota, particularly from soil microbes, might be just as important as species diversity in the conservation of ecosystems and biodiversity. The Soil FungiLog procedure was used to assess functional diversity of soil fungi in a Georgia forest disturbed by human activity and along an elevational gradient in the Chihuahuan Desert. Sites within each location were separated on the basis of fungal carbon substrate utilization profiles. These profiles were analyzed to provide information regarding the functional diversity of soil fungal assemblages at each site. The effects of disturbance and elevation were evaluated with respect to soil fungal functional diversity.     

Give me a sample of air and I will tell which species are found from your region: Molecular identification of fungi from airborne spore samples

Fungi are a megadiverse group of organisms, they play major roles in ecosystem functioning and are important for human health, food production and nature conservation. Our knowledge on fungal diversity and fungal ecology is however still very limited, in part because surveying and identifying fungi is time demanding and requires expert knowledge. We present a method that allows anyone to generate a list of fungal species likely to occur in a region of interest, with minimal effort and without requiring taxonomical expertise. The method consists of using a cyclone sampler to acquire fungal spores directly from the air to an Eppendorf tube, and applying DNA barcoding with probabilistic species identification to generate a list of species from the sample. We tested the feasibility of the method by acquiring replicate air samples from different geographical regions within Finland. Our results show that air sampling is adequate for regional‐level surveys, with samples collected >100 km apart varying but samples collected <10 km apart not varying in their species composition. The data show marked phenology, and thus obtaining a representative species list requires aerial sampling that covers the entire fruiting season. In sum, aerial sampling combined with probabilistic molecular species identification offers a highly effective method for generating a species list of air‐dispersing fungi. The method presented here has the potential to revolutionize fungal surveys, as it provides a highly cost‐efficient way to include fungi as a part of large‐scale biodiversity assessments and monitoring programs.     

A genomic perspective on the shortcomings of mitochondrial DNA for "barcoding" identification

Approximately 600-bp sequences of mitochondrial DNA (mtDNA) have been designated as "DNA barcodes" and have become one of the most contentious and animated issues in the application of genetic information to global biodiversity assessment and species identification. Advocates of DNA barcodes have received extensive attention and promotion in many popular and refereed scientific publications. However, we suggest that the utility of barcodes is suspect and vulnerable to technical challenges that are particularly pertinent to mtDNA. We review the natural history of mtDNA and discuss problems for barcoding which are particularly associated with mtDNA and inheritance, including reduced effective population size, maternal inheritance, recombination, inconsistent mutation rate, heteroplasmy, and compounding evolutionary processes. The aforementioned could significantly limit the application and utility of mtDNA barcoding efforts. Furthermore, global use of barcodes will require application and acceptance of a barcode-based species concept that has not been evaluated in the context of the extensive literature concerning species designation. Implementation of mtDNA barcodes in spite of technical and practical shortcomings we discuss may degrade the longstanding synthesis of genetic and organism-based research and will not advance studies ranging from genomic evolution to biodiversity assessment.     

Two New Computational Methods for Universal DNA Barcoding: A Benchmark Using Barcode Sequences of Bacteria,Archaea, Animals,Fungi, and Land Plants

Taxonomic identification of biological specimens based on DNA sequence information (a.k.a. DNA barcoding) is becoming increasingly common in biodiversity science. Although several methods have been proposed, many of them are not universally applicable due to the need for prerequisite phylogenetic/machine-learning analyses, the need for huge computational resources, or the lack of a firm theoretical background. Here, we propose two new computational methods of DNA barcoding and show a benchmark for bacterial/archeal 16S, animal COX1, fungal internal transcribed spacer, and three plant chloroplast (rbcL, matK, and trnH-psbA) barcode loci that can be used to compare the performance of existing and new methods. The benchmark was performed under two alternative situations: query sequences were available in the corresponding reference sequence databases in one, but were not available in the other. In the former situation, the commonly used “1-nearest-neighbor” (1-NN) method, which assigns the taxonomic information of the most similar sequences in a reference database (i.e., BLAST-top-hit reference sequence) to a query, displays the highest rate and highest precision of successful taxonomic identification. However, in the latter situation, the 1-NN method produced extremely high rates of misidentification for all the barcode loci examined. In contrast, one of our new methods, the query-centric auto-k-nearest-neighbor (QCauto) method, consistently produced low rates of misidentification for all the loci examined in both situations. These results indicate that the 1-NN method is most suitable if the reference sequences of all potentially observable species are available in databases; otherwise, the QCauto method returns the most reliable identification results. The benchmark results also indicated that the taxon coverage of reference sequences is far from complete for genus or species level identification in all the barcode loci examined. Therefore, we need to accelerate the registration of reference barcode sequences to apply high-throughput DNA barcoding to genus or species level identification in biodiversity research.     

DNA barcoding: how it complements taxonomy, molecular phylogenetics and population genetics

DNA barcoding aims to provide an efficient method for species-level identifications and, as such, will contribute powerfully to taxonomic and biodiversity research. As the number of DNA barcode sequences accumulates, however, these data will also provide a unique 'horizontal' genomics perspective with broad implications. For example, here we compare the goals and methods of DNA barcoding with those of molecular phylogenetics and population genetics, and suggest that DNA barcoding can complement current research in these areas by providing background information that will be helpful in the selection of taxa for further analyses.     

Choosing and using a plant DNA barcode

The main aim of DNA barcoding is to establish a shared community resource of DNA sequences that can be used for organismal identification and taxonomic clarification. This approach was successfully pioneered in animals using a portion of the cytochrome oxidase 1 (CO1) mitochondrial gene. In plants, establishing a standardized DNA barcoding system has been more challenging. In this paper, we review the process of selecting and refining a plant barcode; evaluate the factors which influence the discriminatory power of the approach; describe some early applications of plant barcoding and summarise major emerging projects; and outline tool development that will be necessary for plant DNA barcoding to advance.     

Cryptic diversity and significant cophylogenetic signal detected by DNA barcoding the rust fungi (Pucciniaceae) of Cyperaceae–Juncaceae

Plants play important roles as habitat and food for a tremendous diversity of specialist animals and fungi. The disappearance of any plant species can lead to extinction cascades of its associated biota. In consequence, documenting the diversity and specificity of plant-associated organisms is of high practical relevance in biodiversity conservation. Here, we present the first large-scale molecular investigation into the diversity, host specificity, and cophylogenetic congruence of an especially rich plant–fungal association, the rust fungi (Pucciniaceae) of Cyperaceae and Juncaceae. Using the largest rust fungi DNA barcoding dataset published to date (252 sequences, 82 taxa), we reject the presence of a global ITS2-28S barcode gap, but find a local gap in Cyperaceae–Juncaceae rusts, and suggest the existence of many cryptic species in North America, with some broadly circumscribed species possibly corresponding to >10 cryptic species. We test previous hypotheses of correlations between the phylogenies of rust fungi and their Cyperaceae–Juncaceae hosts using a combination of global-fit and event-based cophylogenetic methods. A significant cophylogenetic signal is detected between rusts and their hosts, but the small number of cospeciations argues for preferential host jumps as the driving process behind these correlations. In addition, temporal congruence between the origin of major Carex clades and their rusts suggests that host diversification may have promoted parasite diversification. Finally, we discuss the relevance of rust infection patterns to the systematics of Cyperaceae, highlight some taxonomic problems uncovered by the analyses, and call attention to the promise of DNA barcoding for bridging knowledge gaps in poorly studied plant-associated microorganisms.     

Alpine lichen diversity in an isolated sky island in the Colorado Plateau,USA—Insight from an integrative biodiversity inventory

Lichens are major components of high altitude/latitude ecosystems. However, accurately characterizing their biodiversity is challenging because these regions and habitats are often underexplored, there are numerous poorly known taxonomic groups, and morphological variation in extreme environments can yield conflicting interpretations. Using an iterative taxonomic approach based on over 800 specimens and incorporating both traditional morphology‐based identifications and information from the standard fungal DNA barcoding marker, we compiled a voucher‐based inventory of biodiversity of lichen‐forming fungi in a geographically limited and vulnerable alpine community in an isolated sky island in the Colorado Plateau, USA—the La Sal Mountains. We used the newly proposed Assemble Species by Automatic Partitioning (ASAP) approach to empirically delimit candidate species‐level lineages from family‐level multiple sequence alignments. Specimens comprising DNA‐based candidate species were evaluated using traditional taxonomically diagnostic phenotypic characters to identify specimens to integrative species hypotheses and link these, where possible, to currently described species. Despite the limited alpine habitat (ca. 3,250 ha), we document the most diverse alpine lichen community known to date from the southern Rocky Mountains, with up to 240 candidate species/species‐level lineages of lichen‐forming fungi. 139 species were inferred using integrative taxonomy, plus an additional 52 candidate species within 29 different putative species complexes. Over 68% of sequences could not be assigned to species‐level rank with statistical confidence, corroborating the limited utility of current sequence repositories for species‐level DNA barcoding of lichen‐forming fungi. By integrating vouchered specimens, DNA sequence data, and photographic documentation, we provide an important baseline of lichen‐forming fungal diversity for the limited alpine habitat in the Colorado Plateau. These data provide an important resource for subsequent research in the ecology and evolution of lichens alpine habitats, including DNA barcodes for most putative species/species‐level lineages occurring in the La Sal Mountains, and vouchered collections representing any potentially undescribed species that can be used for future taxonomic studies.     

Analysis of the Protein Domain and Domain Architecture Content in Fungi and Its Application in the Search of New Antifungal Targets

Over the past several years fungal infections have shown an increasing incidence in the susceptible population, and caused high mortality rates. In parallel, multi-resistant fungi are emerging in human infections. Therefore, the identification of new potential antifungal targets is a priority. The first task of this study was to analyse the protein domain and domain architecture content of the 137 fungal proteomes (corresponding to 111 species) available in UniProtKB (UniProt KnowledgeBase) by January 2013. The resulting list of core and exclusive domain and domain architectures is provided in this paper. It delineates the different levels of fungal taxonomic classification: phylum, subphylum, order, genus and species. The analysis highlighted Aspergillus as the most diverse genus in terms of exclusive domain content. In addition, we also investigated which domains could be considered promiscuous in the different organisms. As an application of this analysis, we explored three different ways to detect potential targets for antifungal drugs. First, we compared the domain and domain architecture content of the human and fungal proteomes, and identified those domains and domain architectures only present in fungi. Secondly, we looked for information regarding fungal pathways in public repositories, where proteins containing promiscuous domains could be involved. Three pathways were identified as a result: lovastatin biosynthesis, xylan degradation and biosynthesis of siroheme. Finally, we classified a subset of the studied fungi in five groups depending on their occurrence in clinical samples. We then looked for exclusive domains in the groups that were more relevant clinically and determined which of them had the potential to bind small molecules. Overall, this study provides a comprehensive analysis of the available fungal proteomes and shows three approaches that can be used as a first step in the detection of new antifungal targets.     

Unexpectedly High Levels of Cryptic Diversity Uncovered by a Complete DNA Barcoding of Reptiles of the Socotra Archipelago

Few DNA barcoding studies of squamate reptiles have been conducted. Due to the significance of the Socotra Archipelago (a UNESCO Natural World Heritage site and a biodiversity hotspot) and the conservation interest of its reptile fauna (94% endemics), we performed the most comprehensive DNA barcoding study on an island group to date to test its applicability to specimen identification and species discovery. Reptiles constitute Socotra’s most important vertebrate fauna, yet their taxonomy remains under-studied. We successfully DNA-barcoded 380 individuals of all 31 presently recognized species. The specimen identification success rate is moderate to high, and almost all species presented local barcoding gaps. The unexpected high levels of intra-specific variability found within some species suggest cryptic diversity. Species richness may be under-estimated by 13.8–54.4%. This has implications in the species’ ranges and conservation status that should be considered for conservation planning. Other phylogenetic studies using mitochondrial and nuclear markers are congruent with our results. We conclude that, despite its reduced length (663 base pairs), cytochrome c oxidase 1, COI, is very useful for specimen identification and for detecting intra-specific diversity, and has a good phylogenetic signal. We recommend DNA barcoding to be applied to other biodiversity hotspots for quickly and cost-efficiently flagging species discovery, preferentially incorporated into an integrative taxonomic framework.     

ITS, OTUs and beyond—fungal hyperdiversity calls for supplementary solutions

Molecular species recognition of fungi emerged years before DNA barcoding ( Seifert 2009 ). While the ideal fungal DNA barcode seems Utopian, two research decades nevertheless highlight the internal transcribed spacer (ITS) as the best available choice ( Seifert 2009 ). Databases providing reliable ITS sequences of known fungi require enormous efforts, but are urgently needed ( Abarenkov et al. 2010a,b ; Begerow et al. 2010 ). Any criticism of such a commitment seems unjustified. However, exclusive focus on the development of ITS reference libraries will delay the progress towards a deeper ecological insight. It is widely acknowledged that ITS fails to recognize species, particularly in some ascomycete lineages ( Balajee et al. 2009 ; Seifert 2009 ). It also appears paradoxical to solely rely on ITS for ecological recognition of fungal species when modern fungal systematics rely on phylogenetic species recognition with concordance of multiple gene genealogies (see Blackwell 2011 ). Considering that at least 98% of the predicted ～5 million fungal species remain undescribed ( Blackwell 2011 ), how will reliance on ITS alone influence the biodiversity estimates and ecological understanding? In this issue, Gazis et al. (2011) elegantly demonstrate through multi‐locus sequence phylogeny analyses that ITS largely underestimates the species diversity of tropical fungal endophytes and even more importantly obscures fundamental ecological and biogeographical patterns. This thorough reflection on species delimitation criteria and their implications for ecological and biogeographical inferences underline that ITS, particularly in hyperdiverse habitats, provides no shortcut to deeper knowledge of fungal ecology.     

DNA条形码参考数据集构建和序列分析相关的新兴技术

近年来DNA条形码技术迅速发展, 产生的条形码的数量及其应用范围都呈指数性增长, 现已广泛用于物种鉴定、食性分析、生物多样性评估等方面。本文重点总结并讨论了构建条形码参考数据库和序列聚类相关的信息分析的技术和方法, 包括: 基于高通量测序(high throughput sequencing, HTS)平台以高效并较低的成本获取条形码序列的方法; 同时还介绍了从原始测序序列到分类操作单元(operational taxonomic units, OTUs)过程中的一些计算逻辑以及被广泛采用的软件和技术。这是一个较新并快速发展的领域, 我们希望本文能为读者提供一个梗概, 了解DNA条形码技术在生物多样性研究应用中的方法和手段。     

Diversity and ecology of soil fungal communities in rubber plantations

Monoculture rubber cultivation and its intensive associated human activities are known to have a negative impact on the biodiversity, ecology, and biological conservation of the ecosystems in which they occur. These negative impacts include changes to the biodiversity and function of soil fungal communities, which contribute towards nutrient cycling and interact with other organisms in belowground ecosystems, and may be pathogens. Despite the important role of soil fungi in rubber plantations, these communities have been poorly studied. In this paper, we review the existing literature on the diversity and ecology of belowground fungi in rubber plantations. Various groups of soil fungi, including saprobes, symbionts, and pathogens are discussed. Additionally, the role of plantation management is discussed in the context of both pathogenic soil fungi and the promotion of beneficial soil fungi. Management practices include clone selection, tree age and planting density, application of chemicals, and intercropping systems. Our review shows the strong need for further research into the effects of monoculture rubber plantations on soil fungal communities, and how we can best manage these systems in the future, in order to create a more sustainable approach to rubber production.     

Consideration of the taxonomy and biodiversity of Australian ectomycorrhizal fungi

Mycorrhiza management in forestry must be predicated on an understanding of fungal biology and ecology. A fundamental building block of the biology and ecology of any organism is accurate identification and an understanding of its relationship to other organisms. The taxonomy of the larger fungi has been largely based on morphological classification of sexual structures but now Taxonomy routinely incorporates mating studies, and biochemical and molecular data. Taxonomy may not revolutionize theories on mycorrhiza but can clarify some of the inconsistencies due to misrepresentation or over-generalizations and inappropriate conclusions drawn from studies with inaccurately identified fungi. To illustrate this, we discuss and example where incorrect fungal names were repeatedly erroneously applied in morphological and physiological research reports on this fungus, e.g. Laccaria laccata. In this case subsequent taxonomic study revealed the reason for the conflicting research results reported for this fungus. We discuss the status of identifying the ectomycorrhizal fungi in various forest communities in Australia and the relationships of this process to assessing their use in forestry. Recent intensive efforts to collect, isolate and identify Australian ectomycorrhizal fungi have revealed an enormous and unique species diversity, e.g., for truffle-like fungi, over 2000 collections from the last five years alone have yielded 2 new families, 24 new genera, and about 184 new species. Nearly 95% of the described and undescribed fungi from Australia are novel, with some 22 genera and 3 families endemic. In most cases the current systematic knowledge of mycorrhizal fungi is inadequate to support clear framework for Australian taxa. This reflects the traditional Northern Hemisphere view of the world, the uniqueness of the Australian fungal flora, and how poorly it is known. For example, the genus Hymenogaster had been widely acknowledged as the most reduced member of the Cortinariaceae. However recent work on Hymenogaster species from the Southern Hemisphere has offered a number of alternative affinities to various species. We also discuss the role proper identification of the organisms involved plays in understanding the ecosystem. Emphasis should be placed on how species diversity equates with physiological and genetic diversity and how a sound taxonomic understanding of species and their systematic position is essential to properly manage them. Accurate taxonomic information will continue to be required as the basis for assessing the role of ectomycorrhizal fungi in sustained ecological development. Of particular significance is the role of ectomycorrhizal fungi in maintenance of plant diversity in natural ecosystems and those disturbed by management. In conclusion, we present some key research areas involving the use of taxonomy that need priority attention.     

从文献情报分析看iFlora 的发展趋势

在网络化、信息化逐渐改变人们学习、认知和生活的背景下,本文检索并分析了与iFlora研究相关的DNA条形码、生物多样性信息库、基因测序技术、移动鉴定设备等研究论文和情报,取得下列结果：（1） 植物DNA条形码的研究对象以及研究领域在不断延伸和扩展,但寻找高分辨率的DNA条码和组合片段仍是研究热点,相关的植物分类学、系统发育与演化、生态学、植物多样性等研究也在快速发展;（2） 生物多样性信息数据库建设爆发式增长,为iFlora的知识积累和扩展奠定了基础;（3） 第三代DNA测序技术的发展,快速测序设备的小型化将成为可能;（4） 物种认知和识别的初级移动设备已经出现;（5） 信息技术与植物科学等研究的结合,促进跨领域的研究合作和产品开发。本文讨论了iFlora研究计划,表明其是未来植物多样性研究的发展趋势。     

Some ‘ant’swers: Application of a layered barcode approach to problems in ant taxonomy

DNA barcoding has emerged as a routine tool in modern taxonomy. Although straightforward, this approach faces new challenges, when applied to difficult situation such as defining cryptic biodiversity. Ants are prime examples for high degrees of cryptic biodiversity due to complex population differentiation, hybridization and speciation processes. Here, we test the DNA barcoding region, cytochrome c oxidase 1 and two supplementary markers, 28S ribosomal DNA and long‐wavelength rhodopsin, commonly used in ant taxonomy, for their potential in a layered, character‐based barcoding approach across different taxonomic levels. Furthermore, we assess performance of the character‐based barcoding approach to determine cryptic species diversity in ants. We found (i) that the barcode potential of a specific genetic marker varied widely among taxonomic levels in ants; (ii) that application of a layered, character‐based barcode for identification of specimens can be a solution to taxonomical challenging groups; (iii) that the character‐based barcoding approach allows us to differentiate specimens even within locations based on pure characters. In summary, (layered) character‐based barcoding offers a reliable alternative for problematic species identification in ants and can be used as a fast and cost‐efficient approach to estimate presence, absence or frequency of cryptic species.