A comprehensive and comparative evaluation of primers for metabarcoding eDNA from fish

Accurate assessments of fish species diversity and community composition are essential for understanding fish ecology and managing conservation. Environmental DNA (eDNA) metabarcoding has become an integrated method for monitoring fish species. The accuracy and efficacy of eDNA metabarcoding relies heavily on the choice of amplification primers. A wide selection of metabarcoding primers for fish has been developed; however, there exists no comprehensive and comparative evaluation of their amplification or taxonomic classification of a rich diversity of fish species, which hinders informed decisions regarding their suitability for different study systems.Here, we reviewed the literature and compiled a list of 22 primer sets for eDNA-based metabarcoding analysis of teleost fish, the performance of which was compared using in silico PCR, followed by in vitro metabarcoding analysis using eDNA from waterbodies in Beijing, which harbour a high number of freshwater fish species.We found that the primers showed considerable differences in the amplified taxonomic ranges and proportions, fish taxa richness, species discrimination power, and fish community compositions, both in silico and in vitro. The number of fish taxa detected from eDNA by the primer sets varied from 0 to 66. Primers targeting the 12S rRNA gene generally detected greater fish diversity than those targeting the 16S rRNA or COI genes, while primers targeting the cytochrome b gene amplified the fewest fish taxa in vitro.Regarding target genes, 12S primers generally outperformed other primers in terms of amplified fish diversity. The results of in silico PCR and in vitro tests were not always in agreement, suggesting that primer choice for biodiversity surveys should not be based solely on in silico evaluation. The use of different primers can qualitatively and quantitatively affect the detected biodiversity, which effect should be considered in experimental design and data interpretation. These results will assist with primer selection for eDNA-based fish surveys, and consequently support conservation of freshwater biodiversity.

精准评估鱼类物种多样性与群落组成，是解析鱼类生态学、开展保护管理工作的核心前提。环境DNA（eDNA）宏条形码（metabarcoding）技术已成为鱼类物种监测的主流集成方法。eDNA宏条形码技术的准确性与效能，高度依赖扩增引物的选择。目前已开发出大量鱼类宏条形码引物，但尚无研究针对丰富多样的鱼类物种，对这些引物的扩增效果与分类鉴定能力开展全面的对比评估，这阻碍了研究者针对不同研究场景科学选择适配引物的决策。本研究通过文献调研，整理出22套用于硬骨鱼（teleost fish）eDNA宏条形码分析的引物集，先通过计算机模拟PCR（in silico PCR）对比其性能，随后采用北京地区淡水鱼类资源丰富的水体eDNA样本开展体外（in vitro）宏条形码实验验证。研究结果显示，无论在计算机模拟还是体外实验中，不同引物的扩增分类范围、类群占比、鱼类类群丰富度、物种鉴定能力以及鱼类群落组成均存在显著差异。各引物集从eDNA中检测到的鱼类类群数量介于0至66之间。靶向12S rRNA基因的引物通常比靶向16S rRNA或COI基因的引物能检测到更高的鱼类多样性；而靶向细胞色素b（cytochrome b）基因的引物在体外实验中扩增得到的鱼类类群数量最少。就靶标基因而言，12S引物在扩增鱼类多样性方面整体优于其他引物。计算机模拟PCR与体外实验的结果并非完全一致，这表明生物多样性调查的引物选择不应仅依赖计算机模拟评估。不同引物的使用会从定性与定量层面影响检测到的生物多样性结果，这一效应需在实验设计与数据解读阶段予以考量。本研究结果可为鱼类eDNA调查的引物选择提供参考，进而助力淡水生物多样性的保护工作。