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。

Fungal diversity in herbivore feces in the Tibetan Plateau

The aim of this study is to analyze the diversity of aerobic and anaerobic fungi in feces of several herbivorous animals in the Tibetan Plateau. Eight fresh fecal samples from three types of ruminants (yak, sheep, and goat) and one monogastric animal (horse) were collected. Clone libraries of the internal transcribed spacer (ITS) region of ribosomal DNA amplified from DNA templates isolated from the fecal samples were constructed. PCR amplifications were performed using universal fungal ITS1/ITS4 primers and Neocallimastigomycota-specific MN100/EminITS4 primers separately. Fungal diversity was determined by phylogenetic analyses. A total of 324 sequences were obtained using ITS1/ITS4 primers. The sequences formed 24 operational taxonomic units (OTUs), belonging to three orders of Ascomycota, two orders of Basidiomycota, Mucorales of Zygomycota and the obligate anaerobic Neocallimastigomycota, respectively. OTUs of Ascomycota were the most dominant members, occupying 80.6% of the sequences obtained; whereas those of Neocallimastigomycota were rare (0.6%). The majority of the OTUs detected are distantly related to the known genera or species of fungi. A total of 661 sequences were obtained using MN100/ EminITS4 primers. The sequences were grouped into 29 OTUs, covering all the described genera in Neocallimastigomycota, including Anaeromyces, Caecomyces, Cyllamyces, Neocallimastix, Orpinomyces, and Piromyces, and three uncultured groups (NG9, NG10, and NG11). Three monocentric genera were found in all ruminant samples with Piromyces being the most abundant member (occupying 37.4% of the OTUs recognized). The sequences from the horse feces were all clustered in the NG9 group, which represents a novel clade in Neocallimastigomycota and may be unique to the Tibetan Plateau. The results show that feces of ruminant animals in the Tibetan plateau harbor highly diversified and specific aerobic and anaerobic fungi, many of which remain to be recognized.