High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples

The kingdom Fungi is estimated to include 1.5 million or more species, playing key roles as decomposers, mutualists, and parasites in every biome on the earth. To comprehensively understand the diversity and ecology of this huge kingdom, DNA barcoding targeting the internal transcribed spacer (ITS) region of the nuclear ribosomal repeat has been regarded as a prerequisite procedure. By extensively surveying ITS sequences in public databases, we designed new ITS primers with improved coverage across diverse taxonomic groups of fungi compared to existing primers. An in silico analysis based on public sequence databases indicated that the newly designed primers matched 99% of ascomycete and basidiomycete ITS taxa (species, subspecies or varieties), causing little taxonomic bias toward either fungal group. Two of the newly designed primers could inhibit the amplification of plant sequences and would enable the selective investigation of fungal communities in mycorrhizal associations, soil, and other types of environmental samples. Optimal PCR conditions for the primers were explored in an in vitro investigation. The new primers developed in this study will provide a basis for ecological studies on the diversity and community structures of fungi in the era of massive DNA sequencing.     

Morphological and genetic evidence of contemporary intersectional hybridisation in Mediterranean Helichrysum (Asteraceae,Gnaphalieae)

Hybridisation is considered an important evolutionary phenomenon in Gnaphalieae, but contemporary hybridisation has been little explored within the tribe. Here, hybridisation between Helichrysum orientale and Helichrysum stoechas is studied at two different localities in the islands of Crete and Rhodes (Greece). Using three different types of molecular data (AFLP, nrDNA ITS sequences and cpDNA ndhF sequences) and morphological data, the aim is to provide simultaneous and direct comparisons between molecular and morphological variation among the parental species and the studied hybrid populations. AFLP profiles, ITS sequences and morphological data support the existence of hybrids at the two localities studied, shown as morphological and genetic intermediates between the parental species. Chloroplast DNA sequences show that both parental species can act either as pollen donor or as maternal parent. Fertility of hybrids is demonstrated by the viability of seeds produced by hybrids from both localities, and the detection of a backcross specimen to H. orientale. Although there is general congruence of morphological and molecular data, the analysis of morphology and ITS sequences can fail to detect backcross hybrids.     

PCR产物直接测序还是克隆测序?——密叶杉属rDNA ITS序列的测定方法

通过PCR产物直接测序和克隆测序对三种密叶杉属 (Athrotaxis)植物rDNA内转录间隔区 (ITS)及5 .8SrDNA序列进行了测定与分析。实验表明A .selaginoidesrDNA重复序列间的纯合程度很高 ,对PCR产物直接测序就可以测定其ITS区序列。而A .laxifolia、A .cupressoides的ITS1重复序列间的纯合程度较低 ,各重复单位间序列存在插入 /缺失 ,只有对PCR产物进行克隆测序才能确定其序列。A .laxi folia、A .cupressoides的ITS2区尽管也存在多态性 ,但不同重复序列的浓度比较平均 ,对PCR产物直接测序就可确定重复序列间的变异情况。本实验表明尽管是同一属的三种植物 ,但其rDNA重复序列间的纯合程度不同 ,同一植物ITS的不同区域 ,其重复序列间的纯合程度也不同 ,针对不同的ITS片段可采用不同的方法以测定其序列。     

PCR产物直接测序还是克隆测序 密叶杉属rDNA ITS序列的测定方法

通过PCR产物直接测序和克隆测序对三种密叶杉属（Athrotaxis）植物rDNA内转录间隔区(ITS)及5.8 S rDNA序列进行了测定与分析。实验表明A. selaginoides rDNA重复序列间的纯合程度很高, 对PCR产物直接测序就可以测定其ITS区序列。而A. laxifolia、A. cupressoides的ITS1重复序列间的纯合程度较低,各重复单位间序列存在插入/缺失,只有对PCR产物进行克隆测序才能确定其序列。A. laxifolia、A. cupressoides的ITS2区尽管也存在 多态性,但不同重复序列的浓度比较平均,对PCR产物直接测序就可确定重复序列间的变异情况。本实验表明尽管是同一属的三种植物,但其rDNA重复序列间的纯合程度不同,同一植物ITS的不同区域,其重复序列间的纯合程度也不同,针对不同的ITS片段可采用不同的方法以测定其序列。     

ITS sequence variation and concerted evolution in the natural hybrid species Malus toringoides

The internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) is one of the most used molecular characters in plant systematics. Our previous studies based on morphological analysis and ITS sequence variation suggested that Malus toringoides (Rehd.) Hughes is derived from hybridization between M. transitoria (Batal.) Schneid. and M. kansuensis (Batal.) Schneid. To further understand the variation pattern of ITS sequences in M. toringoides, and to elucidate the evolutionary processes that affect ITS sequence variation after hybridization, we sampled 99 accessions from multiple populations of the hybrid and parental species, and then obtained totally 254 ITS sequences by cloning and sequencing. Our ITS variation data demonstrates three outcomes of ITS repeats after hybrid speciation. ～ 27–41% of M. toringoides have only M. transitoria type ITS sequence, ～ 40–70% have M. transitoria type ITS sequence plus one or two chimeric ITS sequences generated by recombination between parental ITS sequences, and six accessions retain both parental type ITS sequences. The plausible evolutionary processes that created the observed ITS variations were inferred to be the joint actions of recombination, concerted evolution, pseudogenization and backcrossing. Our study provides further understandings of the variation model of ITS repeats after hybridization as well as the evolution of M. toringoides after its hybrid speciation.     

The ITS1-5.8S-ITS2 sequence region in the Musaceae: structure, diversity and use in molecular phylogeny

Genes coding for 45S ribosomal RNA are organized in tandem arrays of up to several thousand copies and contain 18S, 5.8S and 26S rRNA units separated by internal transcribed spacers ITS1 and ITS2. While the rRNA units are evolutionary conserved, ITS show high level of interspecific divergence and have been used frequently in genetic diversity and phylogenetic studies. In this work we report on the structure and diversity of the ITS region in 87 representatives of the family Musaceae. We provide the first detailed information on ITS sequence diversity in the genus Musa and describe the presence of more than one type of ITS sequence within individual species. Both Sanger sequencing of amplified ITS regions and whole genome 454 sequencing lead to similar phylogenetic inferences. We show that it is necessary to identify putative pseudogenic ITS sequences, which may have negative effect on phylogenetic reconstruction at lower taxonomic levels. Phylogenetic reconstruction based on ITS sequence showed that the genus Musa is divided into two distinct clades--Callimusa and Australimusa and Eumusa and Rhodochlamys. Most of the intraspecific banana hybrids analyzed contain conserved parental ITS sequences, indicating incomplete concerted evolution of rDNA loci. Independent evolution of parental rDNA in hybrids enables determination of genomic constitution of hybrids using ITS. The observation of only one type of ITS sequence in some of the presumed interspecific hybrid clones warrants further study to confirm their hybrid origin and to unravel processes leading to evolution of their genomes.     

Species detection using environmental DNA from water samples

The assessment of species distribution is a first critical phase of biodiversity studies and is necessary to many disciplines such as biogeography, conservation biology and ecology. However, several species are difficult to detect, especially during particular time periods or developmental stages, potentially biasing study outcomes. Here we present a novel approach, based on the limited persistence of DNA in the environment, to detect the presence of a species in fresh water. We used specific primers that amplify short mitochondrial DNA sequences to track the presence of a frog (Rana catesbeiana) in controlled environments and natural wetlands. A multi-sampling approach allowed for species detection in all environments where it was present, even at low densities. The reliability of the results was demonstrated by the identification of amplified DNA fragments, using traditional sequencing and parallel pyrosequencing techniques. As the environment can retain the molecular imprint of inhabiting species, our approach allows the reliable detection of secretive organisms in wetlands without direct observation. Combined with massive sequencing and the development of DNA barcodes that enable species identification, this approach opens new perspectives for the assessment of current biodiversity from environmental samples.     

The utility of internally transcribed spacer 2 DNA sequences of the nuclear ribosomal gene for distinguishing sibling species of Trichogramma

The usefulness of the internally transcribed spacer 2 (ITS2) of the nuclear ribosomal gene complex is tested for providing taxonomic characters to identify Trichogramma species. The ITS2 sequences of a group of sibling species of the T. deion/T. pretiosum complexes were determined. A simple and precise identification key to the species of these assemblages was constructed using as taxonomic characters the size of the ITS2 and the difference in restriction length polymorphism of species with similarly sized ITS2. Individual wasps can be identified by amplification of their ITS2 with general primers, determining the size of the PCR product using standard agarose electrophoresis, followed in some species by a DNA-digestion with a restriction enzyme. Because this system works well for a number of closely related species we are hopeful that similar PCR-based identification can be extended to all species of the genus once their ITS2 sequences have been determined. The advantage of this identification system over the morphology-based system is that non-specialists are able to quickly and cheaply identify individual specimens. In addition, species specific primers were tested for the two most common species of these groups (i.e. T. pretiosum and T. deion). These primers can be used either as a direct identification tool or as a method to confirm the identification using the general key. The phylogeny of this group of wasps was also analyzed based on the ITS2 sequence.     

Fungal Community Analysis by Large-Scale Sequencing of Environmental Samples

Fungi are an important and diverse component of soil communities, but these communities have proven difficult to study in conventional biotic surveys. We evaluated soil fungal diversity at two sites in a temperate forest using direct isolation of small-subunit and internal transcribed spacer (ITS) rRNA genes by PCR and high-throughput sequencing of cloned fragments. We identified 412 sequence types from 863 fungal ITS sequences, as well as 112 ITS sequences from other eukaryotic microorganisms. Equal proportions of Basidiomycota and Ascomycota sequences were present in both the ITS and small-subunit libraries, while members of other fungal phyla were recovered at much lower frequencies. Many sequences closely matched sequences from mycorrhizal, plant-pathogenic, and saprophytic fungi. Compositional differences were observed among samples from different soil depths, with mycorrhizal species predominating deeper in the soil profile and saprophytic species predominating in the litter layer. Richness was consistently lowest in the deepest soil horizon samples. Comparable levels of fungal richness have been observed following traditional specimen-based collecting and culturing surveys, but only after much more extensive sampling. The high rate at which new sequence types were recovered even after sampling 863 fungal ITS sequences and the dominance of fungi in our libraries relative to other eukaryotes suggest that the abundance and diversity of fungi in forest soils may be much higher than previously hypothesized.     

New specific primers for amplification of the Internal Transcribed Spacer region in Clitellata (Annelida)

Nuclear molecular evidence, for example, the rapidly evolving Internal Transcribed Spacer region (ITS), integrated with maternally inherited (mitochondrial) COI barcodes, has provided new insights into the diversity of clitellate annelids. PCR amplification and sequencing of ITS, however, are often hampered by poor specificity of primers used. Therefore, new clitellate‐specific primers for amplifying the whole ITS region (ITS: 29F/1084R) and a part of it (ITS2: 606F/1082R) were developed on the basis of a collection of previously published ITS sequences with flanking rDNA coding regions. The specificity of these and other ITS primers used for clitellates were then tested in silico by evaluating their mismatches with all assembled and annotated sequences (STD, version r127) from EMBL, and the new primers were also tested in vitro for a taxonomically broad sample of clitellate species (71 specimens representing 11 families). The in silico analyses showed that the newly designed primers have a better performance than the universal ones when amplifying clitellate ITS sequences. In vitro PCR and sequencing using the new primers were successful, in particular, for the 606F/1082R pair, which worked well for 65 of the 71 specimens. Thus, using this pair for amplifying the ITS2 will facilitate further molecular systematic investigation of various clitellates. The other pair (29F/1084R), will be a useful complement to existing ITS primers, when amplifying ITS as a whole.     

Multiple hybrid formation in natural populations: concerted evolution of the internal transcribed spacer of nuclear ribosomal DNA (ITS) in North American Arabis divaricarpa (Brassicaceae)

DNA sequence variation of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA from Arabis holboellii, A. drummondii, and its putative hybrid A. divaricarpa was analyzed to study hybrid speciation in a species system geographically covering nearly the entire North American continent. Based on molecular systematics the investigated species are better combined under the genus Boechera. Multiple intraindividual ITS copies were detected in numerous accessions of A. divaricarpa, and, to a minor extent, in the parental taxa. Comparative phylogenetic analysis demonstrates that reticulate evolution is common. Consequently, concerted evolution of ITS regions resulted in different types of ITS fragments not only in hybrid populations but also in one of the parental taxa, A. holboellii. Hybrid formation often occurred independently at different sites and at different times, which is reflected by ITS copies resampling the original parental sequence variation in different ways. Some biogeographic structuring of genetic diversity is apparent and mirrors postglacial migration routes. Hybridization, reticulation, and apomixis are assumed to be the major forces driving speciation processes in this species complex. Analysis of conserved regions and secondary structures of the ITS region provided no evidence that, in this system, hybrid ITS evolution is predominantly driven in a particular direction. However, two regions in the ITS1 and ITS2, respectively, show higher mutation rates than expected from outgroup comparisons. Strong evidence for the occurrence of apomixis in A. holboellii and A. divaricarpa has come from pollen size measurements and estimations of pollen quality, which favor the hypothesis that A. drummondii served as paternal hybridization partner more frequently than A. holboellii.     

Intra- and interindividual variation in ITS1 of paragonimus westermani (Trematoda: digenea) and related species: implications for phylogenetic studies

We investigated the utility of the ribosomal first internal transcribed spacer (ITS1) for phylogenetic studies on trematodes of the genus Paragonimus. Numerous clones containing ITS1 PCR products were sequenced for P. miyazakii, P. macrorchis, and members of the P. ohirai and P. westermani species complexes. Some additional data were obtained by direct sequencing of PCR products. The ITS1 is composed of three distinct regions: the short 5' end, followed by a tract of approximately 120 nucleotides which occurs a variable number of times in tandem, and the 3' region, which lacks repeats and is referred to as the "post-repeat" fragment. Sequences from all three regions can be aligned among the species studied. Our initial hypothesis, that the post-repeat region would be valuable for phylogenetic studies within the P. westermani complex, was proved wrong. Intraindividual sequence variation in P. westermani was sometimes greater than between individuals of the species complex. In the P. ohirai species complex, however, sequence variation within individuals was minimal. Possible reasons for these observations are discussed. We also wished to determine whether the length variants sequenced were the dominant variants present in Paragonimus species. This was done by probing Southern blots of genomic digests with an ITS1 fragment which lacks repeat sequences. There is generally greater abundance of large variants, with much lower abundance of small variants, such as those sequenced. Differences in ITS1 lengths are attributed largely to differing numbers of repeats, though some exceptions (which are discussed) were found.     

Fungal community analysis by large-scale sequencing of environmental samples

Fungi are an important and diverse component of soil communities, but these communities have proven difficult to study in conventional biotic surveys. We evaluated soil fungal diversity at two sites in a temperate forest using direct isolation of small-subunit and internal transcribed spacer (ITS) rRNA genes by PCR and high-throughput sequencing of cloned fragments. We identified 412 sequence types from 863 fungal ITS sequences, as well as 112 ITS sequences from other eukaryotic microorganisms. Equal proportions of Basidiomycota and Ascomycota sequences were present in both the ITS and small-subunit libraries, while members of other fungal phyla were recovered at much lower frequencies. Many sequences closely matched sequences from mycorrhizal, plant-pathogenic, and saprophytic fungi. Compositional differences were observed among samples from different soil depths, with mycorrhizal species predominating deeper in the soil profile and saprophytic species predominating in the litter layer. Richness was consistently lowest in the deepest soil horizon samples. Comparable levels of fungal richness have been observed following traditional specimen-based collecting and culturing surveys, but only after much more extensive sampling. The high rate at which new sequence types were recovered even after sampling 863 fungal ITS sequences and the dominance of fungi in our libraries relative to other eukaryotes suggest that the abundance and diversity of fungi in forest soils may be much higher than previously hypothesized. All sequences were deposited in GenBank, with accession numbers AY 969316 to AY 970290 for the ITS sequences and AY 969135 to AY 969315 for the SSU sequences.     

Sequencing of the internal transcribed spacer region ITS1 as a molecular tool detecting variation in the Stylosanthes guianensis species complex

 The internal transcribed spacer (ITS) regions 1 and 2 of the ribosomal DNA from Stylosanthes guianensis CIAT 1283 and cv ‘Schofield’ were amplified by polymerase chain reaction using conserved ITS primers from the 18S, 5.8S and 26S ribosomal genes flanking those regions. The entire region of 683 bp long was cloned, and seven clones were sequenced. Comparison of the ITS spacer regions with published DNA sequences of other plant species revealed limited homology only; this was in contrast to their comparison with the 5.8S rDNA sequences. The ITS1 region of 45 S. guianensis accessions was amplified by PCR and sequenced on both strands using the conserved primers ITS2-ITS5. These sequences, ranging from 201 to 204 bp, were aligned to each other to assess intra-specific polymorphism. Within the S. guianensis (Aubl.) Sw. species complex, 11 DNA sequence types could be distinguished based on an insertion/deletion (indel) event and 15 single base-pair substitutions. In 1 of the S. guianensis types, two kinds of ITS1 sequence were observed in each individual, reminiscent of an incomplete homogenization of the repeat structure in this type. Polymorphisms in the sequence of the ITS1 region were used to define molecular markers for S. guianensis on the basis of PCR-restriction fragment length polymorphism and selective PCR. Received: 24 June 1997 / Accepted: 31 October 1997     

Cephalosporium maydis is a distinct species in the Gaeumannomyces-Harpophora species complex

Cephalosporium maydis is an important plant pathogen whose phylogenetic position relative to other fungi has not been established clearly. We compared strains of C. maydis, strains from several other plant-pathogenic Cephalosporium spp. and several possible relatives within the Gaeumannomyces-Harpophora species complex, to which C. maydis has been suggested to belong based on previous preliminary DNA sequence analyses. DNA sequences of the nuclear genes encoding the rDNA ITS region, β-tubulin, histone H3, and MAT-2 support the hypothesis that C. maydis is a distinct taxon within the Gaeumannomyces-Harpophora species complex. Based on amplified fragment length polymorphism (AFLP) profiles, C. maydis also is distinct from the other tested species of Cephalosporium, Phialophora sensu lato and members of Gaeumannomyces-Harpophora species complex, which supports its classification as Harpophora maydis. Oligonucleotide primers for H. maydis were developed that can be used in a PCR diagnostic protocol to rapidly and reliably detect and identify this pathogen. These diagnostic PCR primers will aid the detection of H. maydis in diseased maize because this fungus can be difficult to detect and isolate, and the movement of authentic cultures may be limited by quarantine restrictions.     

Application of ITS sequences of nuclear rDNA in phylogenetic and evolutionary studies of angiosperms

Nuclear rRNA genes (rDNA) in angiosperms are arranged in long tandem repeat ing units, much like those of other higher eukaryotes. Owing to rapid concerted evolution, the repeat units have homogenized or nearly so in most species. The internal transcribed spacer (ITS) of nuclear rDNA is composed of ITS1 and ITS2, which are seperated by 5.8S rDNA. The two spacers, ITS1 (187～298 bp) and ITS2 (187～252 bp), can be readily amplified by PCR and sequenced using universal primers. The sequences contain many vari able sites and potential informative sites among related species, and have been proven to be a useful molecular marker in phylogenetic and evolutionary studies of many angiosperm taxa. It can be used not only in classification and phylogenetic inferences at the levels of family, subfamily, tribe, genus and section, but also in reconstruction of reticulate evolution and de tection of the speciation via hybridization and polyploidization. But this region may not be useful for resolving phylogenetic relationships among families or taxa of higher hierarchy ow- ing to the rapid variation of the ITS sequences.     

Molecular and morphological evidence for hybridization between endemic Canary Island Convolvulus

Morphological data and molecular data from the chloroplast trn H- psb A region and nuclear ribosomal ITS region are used to test the hypothesis that the problematic Canary Island endemic Convolvulus floridus var. virgatus is a hybrid between the endemic species C. floridus and C. scoparius . Analysis of mean leaf length and width of 58 individuals indicates that accessions referable to C. floridus var. virgatus are intermediate between the parental taxa in leaf dimensions. Direct sequencing of the ITS region of C. scoparius and C. floridus revealed two species-specific ribotypes distinguished by 10 base differences. Examination of ITS chromatograms for putative hybrids revealed polymorphisms at those sites that are diagnostic between species in all except one putative hybrid. Morphological intermediacy and ITS additivity therefore support the hybrid status of C. floridus var. virgatus . An analysis of intraindividual ITS variation confirmed the co-occurrence of both parental ribotypes in putative hybrids and demonstrated that some hybrid individuals contained chimaeric ITS types. It is proposed that chimaeric ITS types are the result of recombination following backcrossing. Sequencing of the trn H- psb A region revealed four haplotypes. Three were, for the most part, confined to C. floridus and putative hybrids although one C. scoparius accession was also found to share this haplotype. The remaining haploype was only found in C. scoparius and putative hybrid accessions. Patterns of haplotype distribution between parental and hybrid accessions suggest multiple, bidirectional hybridization events between C. floridus and C. scoparius . The nomenclature of the hybrid is discussed and the necessary new name C.  ×  despreauxii is proposed.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 154 , 187–204.     

ITS Polymorphisms Shed Light on Hybrid Evolution in Apomictic Plants: A Case Study on the Ranunculus auricomus Complex

The reconstruction of reticulate evolutionary histories in plants is still a major methodological challenge. Sequences of the ITS nrDNA are a popular marker to analyze hybrid relationships, but variation of this multicopy spacer region is affected by concerted evolution, high intraindividual polymorphism, and shifts in mode of reproduction. The relevance of changes in secondary structure is still under dispute. We aim to shed light on the extent of polymorphism within and between sexual species and their putative natural as well as synthetic hybrid derivatives in the Ranunculus auricomus complex to test morphology-based hypotheses of hybrid origin and parentage of taxa. We employed direct sequencing of ITS nrDNA from 68 individuals representing three sexuals, their synthetic hybrids and one sympatric natural apomict, as well as cloning of ITS copies in four representative individuals, RNA secondary structure analysis, and landmark geometric morphometric analysis on leaves. Phylogenetic network analyses indicate additivity of parental ITS variants in both synthetic and natural hybrids. The triploid synthetic hybrids are genetically much closer to their maternal progenitors, probably due to ploidy dosage effects, although exhibiting a paternal-like leaf morphology. The natural hybrids are genetically and morphologically closer to the putative paternal progenitor species. Secondary structures of ITS1-5.8S-ITS2 were rather conserved in all taxa. The observed similarities in ITS polymorphisms suggest that the natural apomict R. variabilis is an ancient hybrid of the diploid sexual species R. notabilis and the sexual species R. cassubicifolius. The additivity pattern shared by R. variabilis and the synthetic hybrids supports an evolutionary and biogeographical scenario that R. variabilis originated from ancient hybridization. Concerted evolution of ITS copies in R. variabilis is incomplete, probably due to a shift to asexual reproduction. Under the condition of comprehensive inter- and intraspecific sampling, ITS polymorphisms are powerful for elucidating reticulate evolutionary histories.     

The origin of the C-genome and cytoplasm of <Emphasis Type="Italic">Avena</Emphasis> polyploids

The contribution of C-genome diploid species to the evolution of polyploid oats was studied using C-genome ITS-specific primers. SCAR analysis among Avena accessions confirmed the presence of C-genome ITS1-5.8S-ITS2 sequences in the genome of AACC and AACCDD polyploids. In situ hybridization and screening of more than a thousand rRNA clones in Avena polyploid species containing the C-genome revealed substantial C-genome rRNA sequence elimination. C-genome clones sequenced and Maximum Likelihood Parsimony analysis revealed close proximity to Avena ventricosa ITS1-5.8S-ITS2 sequences, providing strong evidence of the latter's active role in the evolution of tetraploid and hexaploid oats. In addition, cloning and sequencing of the chloroplastic trnL intron among the most representative Avena species verified the maternal origin of A-genome for the AACC interspecific hybrid formation, which was the genetic bridge for the establishment of cultivated hexaploid oats.     

青藏高原食草动物粪栖真菌的多样性

分别利用真菌通用引物和厌氧真菌特异引物构建了西藏地区3种反刍动物和1种单胃动物共8份新鲜粪样的ITS克隆文库,以通过系统发育分析解析其中好氧真菌与厌氧真菌的多样性。通用引物ITS文库测得324条真菌序列,分别属于子囊菌门Ascomycota 3个目、担子菌门Basidiomycota 2个目、接合菌门Zygomycota的1个目和严格厌氧的新丽鞭毛菌门Neocallimastigomycota,共24个OTUs。其中,子囊菌门相对丰度最高,占80.6%;新丽鞭毛菌门相对丰度最低,仅占0.6%。大部分OTUs与已知真菌属、种关系较远。厌氧真菌特异引物文库测得661条序列,全部属于新丽鞭毛菌门,包括所有已知的厌氧霉属Anaeromyces、盲肠菌属Caecomyces、肠霉属Cyllamyces、新丽鞭毛属Neocallimastix、奥式霉属Orpinomyces、胃梨囊霉属Piromyces 6个属和3个未培养的属级类群(NG9、NG10、NG11),共29个OTUs。其中3个已知的单中心属存在于所有反刍动物样品中,并以Piromyces相对丰度最高(37.4%)。单胃动物马粪样中全部为NG9类群。NG9是本研究新发现的属级类群,研究中同时揭示有多个未培养种和潜在的新种。研究结果证明青藏高原反刍动物粪栖真菌多样性较高,并存在丰富的未培养种和潜在的新属及新种。