Markmann Melanie, Tautz Diethard
Zoological Institute, University of Munich, Luissentrasse 14, 80333 Munich, Germany.
Philos Trans R Soc Lond B Biol Sci. 2005 Oct 29;360(1462):1917-24. doi: 10.1098/rstb.2005.1723.
Organisms living in or on the sediment layer of water bodies constitute the benthos fauna, which is known to harbour a large number of species of diverse taxonomic groups. The benthos plays a significant role in the nutrient cycle and it is, therefore, of high ecological relevance. Here, we have explored a DNA-taxonomic approach to access the meiobenthic organismic diversity, by focusing on obtaining signature sequences from a part of the large ribosomal subunit rRNA (28S), the D3-D5 region. To obtain a broad representation of taxa, benthos samples were taken from 12 lakes in Germany, representing different ecological conditions. In a first approach, we have extracted whole DNA from these samples, amplified the respective fragment by PCR, cloned the fragments and sequenced individual clones. However, we found a relatively large number of recombinant clones that must be considered PCR artefacts. In a second approach we have, therefore, directly sequenced PCR fragments that were obtained from DNA extracts of randomly picked individual organisms. In total, we have obtained 264 new unique sequences, which can be readily placed into taxon groups, based on phylogenetic comparison with currently available database sequences. The group with the highest taxon abundance were nematodes and protozoa, followed by chironomids. However, we find also that we have by far not exhausted the diversity of organisms in the samples. Still, our data provide a framework within which a meiobenthos DNA signature sequence database can be constructed, that will allow to develop the necessary techniques for studying taxon diversity in the context of ecological analysis. Since many taxa in our analysis are initially only identified via their signature sequences, but not yet their morphology, we propose to call this approach 'reverse taxonomy'.
生活在水体沉积层内或之上的生物构成了底栖动物区系，已知其中蕴藏着大量不同分类群的物种。底栖生物在营养循环中发挥着重要作用，因此具有很高的生态相关性。在这里，我们探索了一种DNA分类方法来获取小型底栖生物的物种多样性，重点是从大型核糖体亚基rRNA（28S）的D3 - D5区域获取特征序列。为了广泛代表各类群，我们从德国的12个湖泊采集了底栖生物样本，这些湖泊代表了不同的生态条件。在第一种方法中，我们从这些样本中提取了全基因组DNA，通过PCR扩增相应片段，克隆片段并对单个克隆进行测序。然而，我们发现了相对大量的重组克隆，这些克隆必须被视为PCR假象。因此，在第二种方法中，我们直接对从随机挑选的单个生物体的DNA提取物中获得的PCR片段进行测序。总共，我们获得了264个新的独特序列，基于与当前可用数据库序列的系统发育比较，这些序列可以很容易地归入分类群。分类群丰度最高的是线虫和原生动物，其次是摇蚊。然而，我们也发现，到目前为止，我们尚未穷尽样本中生物的多样性。尽管如此，我们的数据提供了一个框架，在此框架内可以构建一个小型底栖生物DNA特征序列数据库，这将有助于开发在生态分析背景下研究分类群多样性所需的技术。由于我们分析中的许多分类群最初仅通过其特征序列而非形态学来识别，我们建议将这种方法称为“反向分类法”。
