基于高通量测序的水生丝孢菌多样性和分子系统学研究

Environmental DNA were sequenced using High-throughput sequencing to reveal fungal diversity has been carried out in the world, but sequence annotation is challenged because of lack of reference sequence. At the same time, in the face of large numbers of fungi, the way that describe new species based on specimen can’t fully reveal the diversity of fungi, so it is one of the most important tasks in the field of fungal taxonomy to describe new species based on sequences and supply the sequences of the described species. The aquatic hyphomycetes are a key factor in driving carbon cycling and nutrient flow in streams, and they are also an important indicator in testing whether the ecological function of the streams is good. Past survey of diversity based on morphology underestimated its diversity and could not determine the status of molecular systematics. In this project, morphological and molecular technique will be used to study on the diversity of aquatic hyphomycetes in hot spot of biology diversity of China: 1)combining next generation sequencing platforms and PacBio Single Molecule Real-Time sequencing platform, diversity and phylogenetic placement of aquatic hyphomycetes will be uncovered;2) Molecular barcoding of aquatic hyphomycetes which can be cultured will be added and supplemented;3)multiple loci to determine the phylogenetic position of cultured species will be determined by analyzing multigenes.. This study will contribute to establishing of sequence based classification and identification system (SBCI) for fungi, and expected results are great significance for the taxonomy and ecology of fungi.

对环境DNA进行高通量测序揭示真菌多样性已在全球开展，但参考序列缺乏导致序列注释受到挑战。同时，面对宠大的真菌数量，基于标本描述新种的方法不能全面揭示真菌的多样性。因此，基于序列描述新种，补充已描述物种的序列成为真菌分类学将来的主要任务之一。水生丝孢菌是溪流中驱动碳循环和营养流动的关键因子，是溪流生态功能是否完整的重要标志。过去对其多样性调查主要是基于形态学，且分子系统学位置也没深入研究，本申请拟综合形态学和分子生物学技术，针对性地开展中国生物多样性热点地区水生丝孢菌多样性的研究:1)结合二代和三代高通量测序平台，揭示水生丝孢菌的物种多样性和系统学信息;2)提供和补充可培养种的分子条形码，增添条形码数据;3)联合多基因位点确定可培养种的系统学位置。. 本研究有助于构建真菌基于序列的分类和鉴定体系(SBCI),预期结果对真菌分类和生态学也具有重要意义。

水生丝孢菌是栖息在水中腐叶上的一个特殊生态群，在水中腐叶的降解时起着关键作用。本项目在西南地区和海南、广西、广州省多个自然保护区内，共采集腐叶800余份，采用富集培养后单孢挑取的方法，分离到菌株500余株。除已知种外，目前发表新目Neodactylariales,新科Neodactylariaceae各一个, 新属7个： Anisospadicoides, Blastosporium, Phialolunulospora, Antidactylaria, Isthmomyces, Keqinzhangia, Pseudocoronospora, 新种62个，新组合两个。新目和多个新属的相继出现，说明水生丝孢菌形态的多样性及系统学位置的新颖性。. 联合多基因分析，将Keqinzhangia, Pseudocoronospora, Antidactylaria, Isthmomyces四个新属并入座囊菌纲小盾壳科，对Isthmolongispora Matsush.进行了修订。首次将火箭孢属Triscelophorus并入小盾壳科。在上述基础上，发表小盾壳科新种八个，极大地增加了小盾壳科无性态的多样性。. 通过分子系统学分析，确定了Phalangispora, Setosynnema, Uncispora, Seychellomyces, Classicula, Neodactylaria等属的系统学位置,同时提供了多个种的分子条形码。. 高通量测序技术揭示水生腐叶上真菌多样性的结果相比，可培养法能培养出的一些属种，除了Flagellospora外，其他在免培养中都没有检测出来，说明了单孢挑取法分离水生丝孢菌时主要挑到了那些孢子较大和形态特殊的水生丝孢菌，而它们的丰度相对较低，而免培养法检测到的主要是丰度相对高的属，它们是非典型的水生丝孢菌，部分属和陆生环境的重叠。总体而言，在腐叶的降解过程中，并非主要是形态特殊的水生丝孢菌起作用，而是有一些陆生环境也有分布的真菌也参与了降解过程。而认为是水生丝孢菌为何只在水中腐叶上出现，到底是环境选择的结果，还是在陆生环境中丰度较低，用常规方法难以培养，还需进一步探索。