榕小蜂COI基因异质性对DNA条形码鉴定可靠性的影响

During the past decade, DNA barcoding has developed as a hotspot in the subject of biology. Though the phenomenon of mitochondrial heteroplasmy- more than one haplotypes coexist within an individual- may have negative effect on the identification efficacy of DNA barcoding, no study has been demonstrated to test the effect by next-generation sequencing technology, or to investigate the evolutionary pattern of mitochondrial heteroplasmy in diverse species. Our previous study based on the insect group of fig wasps- the various chalcidoid species living in the fig syconia, which present diverse biological habits and display extreme morphological variations - discovered evidence showing that some species have distinct heteroplasmy of mtDNA. In this study, based on the 37 fig wasp species belonging to three families and living on 11 fig species (Ficus: Moraceae), we will demonstrate next-generation sequencing on all the amplicons of DNA barcoding gene COI, to thoroughly investigate the distribution of mitochondrial heteroplasmy in each species, to compare the results with the identification results via standardized barcoding process based on sanger sequencing of the PCR amplicons, to discover the impact of mitochondrial heteroplasmy on the identification efficacy of DNA barcding in diverse species, to unearth the distribution pattern of mitochondrial heteroplasmy among different species, and to further explore the putative correlations between heteroplasmy and the species’ biological or morphological characteristics. This study may help to rationally handle the negative effect of mitochondrial heteroplasmy on the application of DNA barcoding, and to discover the evolutionary pattern and history of mitochondrial heteroplasmy in the fig wasps.

DNA条形码是生物学领域近十多年飞速发展起来的学科热点。人们虽认识到线粒体异质性（多个单体型共存于同一个体）对DNA条形码的鉴定效力会产生负面影响，然而还没人运用二代测序技术研究条形码基因异质性对鉴定可靠性的影响，更没有线粒体异质性分布规律等相关进化研究。聚集生活在榕果中的小蜂（统称榕小蜂），生物习性和种类多样，形态变异剧烈。已有证据发现部分种类具有极高的线粒体基因异质性。本项目选用来自11种榕树、分属３个科的37种小蜂，利用二代测序技术对DNA条形码COI基因进行扩增子测序，全面调查物种的异质性分布，同时与常规DNA条形码的PCR产物直接测序或克隆测序的鉴定结果比较，发现异质性对鉴定结果的影响程度，发掘异质性在物种间的分布规律，探索其与物种可能的生物学和形态学联系。本研究将有助于在DNA条形码的鉴定过程中正确处理线粒体基因异质性的负面影响，也有助于了解榕小蜂线粒体异质性的进化格局和历程。

DNA条形码是生物学领域近十多年飞速发展起来的学科热点。人们虽认识到线粒体异质性（多个单体型共存于同一个体）对DNA条形码的鉴定效力会产生负面影响，然而还没人运用二代测序技术研究条形码基因异质性对鉴定可靠性的影响，更没有线粒体异质性分布规律等相关进化研究。本项目选用了28种榕小蜂物种的雌雄样本一共155头个体对DNA条形码序列区域进行Sanger直接测序、克隆测序、以及高通量扩增子测序，针对这些数据的分析结果表明线粒体异质性会导致基于DNA条形码、尤其是DNA宏条形码技术的榕小蜂物种鉴定数量高估。进一步扩大样本范围，针对蛛形纲以及昆虫纲90个物种的研究结果也同样显示，线粒体异质性会导致基于DNA宏条形码技术评估生物多样性的结果被高估，不仅在榕小蜂类群里如此，在整个昆虫纲以及蛛形纲里也同样如此。鉴于DNA条形码以及DNA宏条形码技术被广泛用于这些类群的物种鉴定和物种多样性评估，我们的研究结果将给被广泛使用的DNA宏条形码技术提供一些冷静思考，对DNA条形码技术的发展提供不同视角的审视。