鱼类环境DNA metabarcoding片段的近缘物种识别差异

已知的鱼类环境DNA（eDNA）metabarcoding片段均未被针对性考察其对近缘物种的适用性，实际使用过程中存在“物种丢失”风险。为筛选出物种识别率最高的片段，本研究比较了15个主流片段对106属（共935种）鱼类的识别差异。研究结果如下：（1）蛋白质编码基因（COI，片段15）的物种识别率最高，但其引物通用性最差；片段09、片段11、片段07、片段03、片段12的引物序列总平均遗传距离也较大，均存在eDNA低效扩增的风险；（2）片段长度影响物种识别率，核糖体基因中片段05、片段06、片段01、片段02及片段13的物种识别率较高；（3）非度量多维尺度分析（NMDS）显示，不同基因、同一基因不同片段的识别结果存在较大差异，应考虑多片段、多基因组合应用；片段01与片段02、片段05与片段06等在NMDS图上距离较近，存在相互替代性；（4）物种类群影响识别结果，eDNA研究仍需要进一步开发高识别率片段。综合物种识别率、引物通用性、NMDS分析等多方面因素，本研究推荐2×150 bp测序平台使用片段01（MiFish-U）、2×250 bp测序平台使用片段05（Ac12S），辅以片段13（Vert-16S-eDNA）等进行近缘鱼类多样性调查。本研究旨在为提高鱼类eDNA调查结果准确性提供一定技术支撑。

Differences of eDNA metabarcoding fragments in relative fish species resolution

The applicability of environmental DNA (eDNA) metabarcoding fragments to relative fish species had not been compared. There was a risk of “species loss” in diversity surveys. In order to screen out the best fragments, we compared the resolution rate differences of 15 eDNA metabarcoding fragments in 106 genera (a total of 935 species). The results were as follows: (1) the protein-coding gene (COI, fragment 15) had the highest resolution rate, but the universality of its corresponding primer pairs was the worst; the overall mean distance based on primer pair sequence of fragment 09, fragment 11, fragment 07, fragment 03 and fragment 12 were obviously large, suggesting their eDNA amplification efficiency were possibly low. (2) The resolution rates were significantly affected by the length of fragments, and the fragment 05, fragment 06, fragment 01, fragment 02 and fragment 13 of ribosomal genes had a higher resolution rate except fragment 15. (3) Non-metric multidimensional scaling analysis (NMDS) showed that there were great differences among different genes and different fragments belonging to the same gene. Therefore, the combination application of multi-fragment and multi-gene should be considered; besides, fragment 01 and fragment 02, and fragment 05 and fragment 06 were close to each other in the NMDS plot. They function of fish resolution were overlapped. (4) Species groups affected the resolution results, and eDNA studies stilled need to develop fragments with higher resolution rates. Based on the resolution rate of relative species, the universality of primer pairs and NMDS analysis, this study recommended fragment 01 (Mifish-U) for 2×150 bp sequencing platform and fragment 05 (Ac12S) for 2×250 bp sequencing platform, supplemented by fragment 13 (Vert-16S-eDNA) to investigate the diversity of relative fish. This study provided some support for improving the accuracy of fish eDNA survey results.