College of Life Sciences, Nankai University, Tianjin, China.
College of Horticulture and Landscape, Tianjin Agricultural University, Tianjin, China.
Mol Ecol Resour. 2022 Nov;22(8):2967-2980. doi: 10.1111/1755-0998.13678. Epub 2022 Jul 5.
DNA barcoding and metabarcoding have been increasingly used in species delimitation and species diversity assessment, respectively, and the molecular markers used in animals are mainly derived from mitochondrial DNA. It is well known that the phenomenon of multiple mitochondrial haplotypes within the same specimen (hereafter referred to as "mitotype diversity") may have a negative impact on the proper assessment of biodiversity by metabarcoding. However, few studies have focused on the incidence of this phenomenon and its effects on metabarcoding results using different sample preparation strategies, such as mock community construction using pooled high-throughput sequencing (HTS) data, DNA-pooling and Tissue-pooling. In this study, we investigated mitotype diversity and its influence on metabarcoding based on 398 specimens from 66 species of Insecta and 82 specimens from 16 species of Arachnida by HTS of the mitochondrial cox1 gene fragment. The results revealed that mitotype diversity was common in the studied taxa and significantly increased the number of operational taxonomic units (OTUs) using the three sample preparation strategies. The results also showed that the bioinformatics pipeline based on authentic amplicon sequence variants was more reliable than the pipeline based on OTUs. Regarding the sample preparation strategies of DNA-pooling and Tissue-pooling commonly used in metabarcoding, our results revealed that their results of metabarcoding were quite similar, and the Tissue-pooling strategy was therefore preferred because of its simplicity. Our study calls for additional attention to the interference of mitotype diversity on the results of DNA metabarcoding in biodiversity assessment.
DNA 条形码和代谢条形码分别越来越多地用于物种划定和物种多样性评估，动物中使用的分子标记主要来自线粒体 DNA。众所周知，同一标本内存在多个线粒体单倍型的现象（以下简称“线粒体多态性”）可能对代谢条形码对生物多样性的正确评估产生负面影响。然而，很少有研究关注这种现象的发生率及其对使用不同样品制备策略（如使用高通量测序（HTS）数据汇集构建模拟群落、DNA 池化和组织池化）的代谢条形码结果的影响。在这项研究中，我们通过 HTS 对来自昆虫纲 66 个物种的 398 个标本和蛛形纲 16 个物种的 82 个标本的线粒体 cox1 基因片段进行了线粒体多态性及其对代谢条形码影响的研究。结果表明，在研究的分类群中，线粒体多态性很常见，并且使用三种样品制备策略显著增加了操作分类单元（OTUs）的数量。结果还表明，基于真实扩增子序列变异的生物信息学管道比基于 OTUs 的管道更可靠。关于代谢条形码中常用的 DNA 池化和组织池化样品制备策略，我们的研究结果表明，它们的代谢条形码结果非常相似，因此，由于其简单性，组织池化策略更受欢迎。我们的研究呼吁在生物多样性评估中，对 DNA 代谢条形码结果中由于线粒体多态性产生的干扰给予更多关注。
