Zenker Mauricio M, Specht Alexandre, Fonseca Vera G
Zoological Research Museum Alexander Koenig Bonn Germany.
Present address: Rua Eulo Maroni São Paulo Brazil.
Ecol Evol. 2020 Feb 25;10(5):2352-2366. doi: 10.1002/ece3.6042. eCollection 2020 Mar.
Automated species identification based on data produced with metabarcoding offers an alternative for assessing biodiversity of bulk insect samples obtained with traps. We used a standard two-step PCR approach to amplify a 313 bp fragment of the barcoding region of the mitochondrial COI gene. The PCR products were sequenced on an Illumina MiSeq platform, and the OTUs production and taxonomic identifications were performed with a customized pipeline and database. The DNA used in the PCR procedures was extracted directly from the preservative ethanol of bulk insect samples obtained with automatic light traps in 12 sampling areas located in different biomes of Brazil, during wet and dry seasons. Agricultural field and forest edge habitats were collected for all sampling areas. A total of 119 insect OTUs and nine additional OTUs assigned to other arthropod taxa were obtained at a ≥97% sequence similarity level. The alpha and beta diversity analyses comparing biomes, habitats, and seasons were mostly inconclusive, except for a significant difference in beta diversity between biomes. In this study, we were able to metabarcode and HTS adult insects from their preservative medium. Notwithstanding, our results underrepresent the true magnitude of insect diversity expected from samples obtained with automatic light traps in Brazil. Although biological and technical factors might have impacted our results, measures to optimize and standardize eDNA HTS should be in place to improve taxonomic coverage of samples of unknown diversity and stored in suboptimal conditions, which is the case of most eDNA samples.
基于元条形码技术产生的数据进行自动物种鉴定，为评估通过陷阱采集的大量昆虫样本的生物多样性提供了一种替代方法。我们采用标准的两步PCR方法扩增线粒体COI基因条形码区域的一个313bp片段。PCR产物在Illumina MiSeq平台上进行测序，并使用定制的流程和数据库进行OTU生成和分类鉴定。PCR程序中使用的DNA直接从巴西不同生物群落的12个采样区域通过自动诱虫灯采集的大量昆虫样本的保存乙醇中提取，在雨季和旱季均进行了采集。所有采样区域都收集了农田和森林边缘栖息地的样本。在序列相似度≥97%的水平上，共获得了119个昆虫OTU和另外9个归属于其他节肢动物类群的OTU。比较生物群落、栖息地和季节的α和β多样性分析大多没有定论，不过生物群落之间的β多样性存在显著差异。在本研究中，我们能够对成年昆虫保存在保存介质中的DNA进行元条形码和高通量测序分析。尽管如此，我们的结果并未充分反映出巴西自动诱虫灯采集样本中预期的昆虫多样性的真实规模。虽然生物学和技术因素可能影响了我们的结果，但应采取措施优化和标准化环境DNA高通量测序，以提高对未知多样性且保存在次优条件下的样本（大多数环境DNA样本都是这种情况）的分类覆盖范围。
