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张顺,林施泉,孟凡旭,吴敏,许学伟,王春生.太平洋克拉里昂-克利伯顿断裂带嘴刺目线虫多样性.生态学报,2017,37(5):1630~1638 本文二维码信息
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太平洋克拉里昂-克利伯顿断裂带嘴刺目线虫多样性
Nematode (Enoplida) diversity in sediment samples collected from the Clarion-Clipperton Fracture Zone
投稿时间:2015-09-18  修订日期:2016-06-21
DOI: 10.5846/stxb201509181920
关键词嘴刺目线虫  多样性  系统发育  克拉里昂-克利伯顿断裂带
Key Wordsenoplida  diversity  phylogeny  Clarion-Clipperton Fracture Zone
基金项目国际海域资源调查与开发“十二五”课题(DY125-14-E-02;DY125-22-QY-29)
作者单位E-mail
张顺 国家海洋局第二海洋研究所, 杭州 310012
浙江大学生命科学学院, 杭州 310058 
 
林施泉 国家海洋局第二海洋研究所, 杭州 310012  
孟凡旭 国家海洋局第二海洋研究所, 杭州 310012  
吴敏 浙江大学生命科学学院, 杭州 310058  
许学伟 国家海洋局第二海洋研究所, 杭州 310012  
王春生 国家海洋局第二海洋研究所, 杭州 310012 wang-sio@163.com 
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摘要:
从太平洋深海克拉里昂-克利伯顿断裂带(Clarion-Clipperton fracture zone,简称CC区)4个站位采集的深海沉积物样品中检出26条嘴刺目(Enoplida)线虫个体。综合应用形态学和分子生物学方法,共鉴定嘴刺目线虫6科8属,其中尖口线虫科(Oxystominidae)个体数量最多,占总数的57.7%,其次为前感线虫科(Anticomidae,19.2%)、光皮线虫科(Phanodermatidae,7.7%)、钩线虫科(Oncholaimidae,7.7%)、烙线虫科(Ironidae,3.8%)和矛线虫科(Enchelidiidae,3.8%)。科、属组成与相邻站点同期采样所获的线虫近似,而丰度组成比例有所差异。分子生物学方法获得了线虫rRNA基因序列16条,经GenBank数据库比对,其与已有的序列相似性范围为94%-99%,以此为依据可确定到科的水平和大部分属的水平(84.6%)。DNA条形码比对结果和形态学鉴定结果有较高一致性,表明分子条形码技术可作为深海线虫鉴定的有效手段。系统发育分析结果显示,基于18S和28S rRNA基因序列,采用不同方法构建系统发育树,其分支结构基本一致;钩线虫科和矛线虫科聚类在一起,光皮线虫科和前感线虫科聚类在一起,显示出彼此间较近的遗传关系。
Abstract:
Marine free-living nematodes (Phylum Nematoda) are widespread and abundant in marine sediments, often representing 70%-90% of the benthic metazoans. However, marine nematode taxonomy is severely underdeveloped, and about only 4,000 species of free-living marine nematodes have been described. Nematode identification by using traditional morphological methods is time consuming and expensive. Some marine nematodes are small, displaying similar morphological characters and are difficult to identify by traditional methods. Molecular technology, or "barcoding," offers the potential of a fast and objective way of species identification. The small and large subunit ribosomal DNA (SSU rDNA and LSU rDNA) gene-based phylogenetic analysis is a powerful tool for clarifying evolutionary relationships among nematode taxa. The order Enoplida is one of the most important groups of marine nematodes. Many enoplids are possibly active predators, and play important ecological roles in marine environments. Here we reported the isolation of 26 nematodes, belonging to Enoplida, from sediment samples collected at four sites in the Clarion-Clipperton Fracture Zone of the Pacific Ocean. The specimens were preserved in DESS solution (20% dimethyl sulphoxide, 0.25 mol/L disodium EDTA pH 8.0, saturated with NaCl) immediately after collection. Each sediment sample was rinsed through a 38-μm sieve using filtered seawater, and extracted using the Ludox flotation method. Nematodes were placed on temporary slides and observed using a Leica DM5500 microscope. After image capturing, each specimen was washed, cut into several pieces, transferred into micro-centrifuge tubes, and digested with Proteinase K. A series of frozen specimens was subsequently thawed and subjected to PCR amplification of the 18S rRNA gene and D3 expansion segments of the 28S rRNA gene. Sequences were analyzed and compared with published data from GenBank by means of a BLAST search. Phylogenetic trees were constructed using the neighbor-joining, maximum likelihood, and maximum parsimony methods with the MEGA5 program package, after multiple alignment of the data by CLUSTAL W. Based on morphological and molecular analyses, these 26 nematodes were classified into six families and eight genera, among which Oxystominidae was the most abundant family, accounting for 57.7%. The other families included Anticomidae (19.2%), Phanodermatidae (7.7%), Oncholaimidae (7.7%), Ironidae (3.85%), and Enchelidiidae (3.85%). At the family and genus level, the community composition at adjacent sites during the same period showed similar results, but the abundance was different. Sixteen sequences of rRNA gene were obtained in the present study and their similarity to the sequences in GenBank ranged from 94 to 99%. According to the results from BLAST, all sequences could be identified at the level of family, while 84.6% of the 16 sequences could be identified at the level of genus. The results from morphological and molecular analysis showed high consistency, which suggested that molecular barcoding is an efficient method to identify deep-sea nematodes. Phylogenetic trees constructed from the sequences of 18S and 28S rRNA gene showed similar topological structures; the species of Oncholaimidae and Enchelidiidae were clustered into one group, whereas those of Phanodermatidae and Anticomidae were clustered into another group, indicating their close genetic relationships.
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