Metabarcoding and mitochondrial metagenomics of endogean arthropods to unveil the mesofauna of the soil
Metabarcoding and mitochondrial metagenomics of endogean arthropods to unveil the mesofauna of the soil
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DOI：
10.1111/2041-210x.12557
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发表时间：
2016-09-01
影响因子：
6.6
通讯作者：
Vogler, Alfried P.
中科院分区：
文献类型：
--
作者：
Arribas, Paula;Andujar, Carmelo;Vogler, Alfried P.
居住在土壤中的生物群落是最多样化、最复杂、但研究最少的陆地生态系统之一。最大的知识差距适用于节肢动物(身体大小为01-2毫米)，因为在许多情况下，传统的形态和分子方法不足以描述这些复杂的群落。高通量测序(HTS)方法的发展需要解决目前的障碍，并进一步促进我们对地下生物多样性的理解。我们提出了一种浮选-Berlese-Flotation(FBF)的采样和样本处理方案，以从土壤中获得节肢动物的清洁DNA提取。此外，我们开发并测试了HTS协议，用于在MiSeq Illumina平台上使用cox1元基因组编码和鸟枪元基因组测序来表征这些批量DNA提取的节肢动物群落。FBF协议从足够大的土壤中提供土壤节肢动物的DNA，并且不受细菌和抑制剂的污染。来自伊比利亚草原的两个深层土壤样品的变生物编码和超基因组测序揭示了28科100种螨类和弹尾目。直接从散装标本的鸟枪测序中组装出54个物种的部分和完整的有丝分裂基因组，平均长度为6000bp。元编码和元基因组测序得到了非常一致的OTU，但元编码的物种数量最高，而大约73%的物种通过与猎枪序列匹配的读数得到确认，通过从这些猎枪读数进行重叠群组装得到的物种数量接近48%。FBF协议与基于聚合酶链式反应的测序管道和鸟枪式测序管道相结合，解决了在MiSeq Illumina平台上研究土壤节肢动物的大部分挑战。它们是在系统发育和群落生态学背景下描述土壤多样性的强大、成本效益高的工具。HTS研究方法的这些方法论发展将促进生态和进化研究、土壤节肢动物的生物监测以及理论和应用土壤科学的进步。
Biological communities inhabiting the soil are among the most diversified, complex and yet most poorly studied terrestrial ecosystems. The greatest knowledge gaps apply to the arthropod mesofauna (01-2mm body size) because conventional morphological and molecular approaches are in many cases insufficient for the characterisation of these complex communities. The development of high-throughput sequencing (HTS) methodologies is required to solve current impediments and to further advance our understanding of below-ground biodiversity. We propose a flotation-Berlese-flotation (FBF) protocol for sampling and specimen processing to obtain clean' DNA extractions of arthropod mesofauna from the soil. In addition, we developed and tested HTS protocols for the characterisation of arthropod communities from these bulk DNA extractions using cox1 metabarcoding and shotgun metagenomic sequencing on the MiSeq Illumina platform. The FBF protocol provided DNA of soil arthropods from sufficiently large volumes of soil and free from contaminating bacteria and inhibitors. Metabarcoding and metagenomic sequencing on two deep soil samples from Iberian grasslands revealed >100 species of Acari and Collembola from 28 families. Genome assembly straight from shotgun sequencing of bulk specimens produced partial and full mitogenomes for 54 species with average length of >6000bp. Metabarcoding and metagenomic sequencing resulted in closely congruent OTUs, but species numbers were highest with metabarcoding, while approximate to 73% of species were confirmed by matching shotgun sequence reads and approximate to 48% by contig assembly from those shotgun reads. In combination, the FBF protocol together with the PCR-based and shotgun sequencing pipelines addressed most of the challenges of studying soil arthropod mesofauna on the MiSeq Illumina platform. They are powerful, cost-efficient tools for characterising soil diversity in a phylogenetic and community ecology context. These methodological developments of HTS approaches for the study of mesofauna will accelerate ecological and evolutionary studies, biomonitoring of soil arthropods, and progress in both theoretical and applied soil science.