Veríssimo Joana, Lopes-Lima Manuel, Amaral Fábio, Chaves Cátia, Fernandes Vasco, Kemanja Mutaleni, Teixeira Amílcar, Martins Filipa M S, Beja Pedro
CIBIO, Centro de Investigação Em Biodiversidade e Recursos Genéticos, InBIO Laboratório Associado, Campus de Vairão, Universidade do Porto, Vairão, Portugal.
BIOPOLIS Program in Genomics, Biodiversity and Land Planning, CIBIO, Campus de Vairão, Vairão, Portugal.
Mol Ecol Resour. 2025 Aug;25(6):e14082. doi: 10.1111/1755-0998.14082. Epub 2025 Apr 1.
Environmental DNA (eDNA) metabarcoding technologies promise significant advances in biodiversity monitoring, yet their application requires extensive optimisation and standardisation. Recent research demonstrated that increased sampling and analytical efforts are needed to improve biodiversity estimates, though fully optimising study designs is often hindered by resource constraints. Consequently, researchers must carefully navigate methodological trade-offs to design effective eDNA metabarcoding monitoring studies. We conducted a water eDNA survey of vertebrates in a Mediterranean watershed to identify key methodological factors influencing species richness and composition estimates. We examined the impacts of using high- versus low-capacity filtration capsules, varying levels of biological and technical replication, and the pooling of PCR replicates before indexing. The primary sources of variation identified were capsule filtration capacity and site replication across the watershed. While biological replication within sites and PCR replication also improved biodiversity estimates, their effects were comparatively smaller. Pooling PCR replicates before indexing performed more poorly than analysing them independently. Methodological impacts were stronger on terrestrial than on aquatic species. Based on these results, we recommend that priority should be given to high-capacity filtration and sampling across multiple sites. Site-level replication deserves lower priority, especially when filtering large water volumes. PCR replication is crucial for detecting rare species but should be balanced with increased site sampling and eventually site-level replication. Avoiding the pooling of PCR replicates is important to enhance sensitivity for rare species. Overall, we stress the importance of balancing methodological choices with resource constraints and monitoring goals, and we emphasise the need for research assessing methodological trade-offs in different study systems.
环境DNA（eDNA）宏条形码技术有望在生物多样性监测方面取得重大进展，但其应用需要广泛的优化和标准化。最近的研究表明，需要增加采样和分析工作以改善生物多样性估计，尽管全面优化研究设计往往受到资源限制的阻碍。因此，研究人员必须谨慎权衡方法，以设计有效的eDNA宏条形码监测研究。我们对地中海流域的脊椎动物进行了一次水体eDNA调查，以确定影响物种丰富度和组成估计的关键方法因素。我们研究了使用高容量与低容量过滤胶囊、不同水平的生物和技术重复以及在索引前合并PCR重复的影响。确定的主要变异来源是胶囊过滤能力和流域内的采样点重复。虽然采样点内的生物重复和PCR重复也改善了生物多样性估计，但其影响相对较小。在索引前合并PCR重复的效果比单独分析它们要差。方法学影响对陆生物种的作用比对水生物种更强。基于这些结果，我们建议应优先考虑高容量过滤和跨多个采样点采样。采样点水平的重复应给予较低优先级，尤其是在过滤大量水体时。PCR重复对于检测稀有物种至关重要，但应与增加采样点采样并最终与采样点水平的重复相平衡。避免合并PCR重复对于提高对稀有物种的敏感性很重要。总体而言，我们强调在方法选择与资源限制和监测目标之间取得平衡的重要性，并强调需要开展研究评估不同研究系统中的方法权衡。
