Metabarcoding versus species-specific primers to estimate <em>Salmo trutta</em> biomass and density in mountain streams

Environmental DNA (eDNA) is a cost-efficient, non-invasive method to monitor fish populations, but the quantitative aspect of this technique (e.g., estimating biomass or densities) remains underexplored. Few studies have established relationships between fish DNA concentration and biomass/density. Here, we investigate the relationship between eDNA concentration (copies per liter) and trout biomass and densities estimated by electrofishing in mountain streams of Picos de Europa National Park (Spain). We assessed eDNA effectiveness in inferring biomass/density using 18S rRNA (18S) and cytochrome c oxidase I (COI) metabarcoding, and quantitative PCR with a COI-specific Salmo trutta primer, each performed with different datasets from the same sampling points. Salmonidae eDNA concentration positively correlates with trout biomass and density. Both 18S and specific-COI markers showed a significant increase in DNA concentration as trout biomass and density rose in electrofishing surveys. However, general COI did not exhibit significant trout DNA concentration and biomass/density relationships, despite providing greater taxonomic resolution at the species level. Further analysis exploring eDNA concentration and biomass/densities across different trout size classes (fry, juvenile, and adult) revealed that juvenile trout biomass contributed the most to the observed eDNA concentration – biomass/density relationship. Our results suggest that DNA concentration estimated from metabarcoding, when using an appropriately selected primer, can reliably indicate trout biomass and density in these mountain streams where trout is the dominant species. Although quantitative PCR showed similar trends, it had lower explanatory power. This study highlights the importance of integrating a quantitative framework in metabarcoding for ecological monitoring and biodiversity assessments. Factors such as amplicon length, genetic region, marker specificity, or fish size class can influence the relationship between sequencing reads and electrofishing data. This methodology could aid the conservation and management of fish populations and other communities, though further research is needed to extend these results and assess eDNA detection reliability.环境DNA（eDNA）是一种经济高效、非侵入式的鱼类种群监测手段，但该技术的定量应用（例如估算生物量或种群密度）仍未得到充分探索。目前鲜有研究建立鱼类DNA浓度与生物量、种群密度之间的定量关联。本研究以西班牙欧洲之峰国家公园的山地溪流为研究区域，通过电渔法估算溪流中褐鳟的生物量与种群密度，探究eDNA浓度（拷贝数/升）与二者的关联。我们基于同一采样点的多组独立数据集，分别采用18S核糖体RNA（18S rRNA）、细胞色素c氧化酶I（COI）宏条形码测序（metabarcoding），以及针对褐鳟COI基因的特异性引物开展定量聚合酶链反应（quantitative PCR）实验，以评估eDNA推断生物量与种群密度的有效性。鲑科鱼类eDNA浓度与褐鳟生物量、种群密度呈显著正相关。在电渔法调查中，18S和特异性COI两种分子标记均显示，随着褐鳟生物量与种群密度升高，eDNA浓度显著上升。不过，通用COI标记虽能提供更高的物种水平分类分辨率，却未呈现出与褐鳟eDNA浓度及生物量、种群密度的显著关联。进一步针对不同体型等级褐鳟（鱼苗、幼鱼、成鱼）的eDNA浓度与生物量、种群密度展开分析后发现，幼鱼生物量对观测到的eDNA浓度-生物量/密度关联贡献最大。本研究结果表明，若选用合适的引物，通过宏条形码测序得到的DNA浓度可可靠指示以褐鳟为优势物种的山地溪流中的褐鳟生物量与种群密度。尽管定量聚合酶链反应也呈现出相似趋势，但其解释力较低。本研究强调了在宏条形码测序中整合定量框架用于生态监测与生物多样性评估的重要性。扩增子长度、遗传区域、标记特异性以及鱼类体型等级等因素，均可影响测序读数与电渔法数据之间的关联。该方法可为鱼类种群及其他生物群落的保护与管理提供助力，但仍需开展进一步研究以拓展上述结论，并评估eDNA检测的可靠性。