1. Environmental DNA (eDNA) from water samples is increasingly used to detect the presence and distribution of species in aquatic ecosystems. However, before implementing eDNA in monitoring programmes, various species-specific sampling or analytical issues remain to be resolved in order to minimize frequencies of false-positive and -negative results. For example, empty shells from freshwater pearl mussels (Margaritifera margaritifera) contain extractable DNA (chemical extraction from ground-up shells) suggesting a risk of false-positive samples at stream sites with extinct populations but with empty shell material remaining.
2. The aim of this study was to investigate whether empty and naturally degrading shells from M. margaritifera can cause false-positive eDNA signals in water samples.
3. Water samples were collected from outdoor stream channels (in Lemming, Denmark) with living freshwater pearl mussels or empty shell material (density ~10 individuals m⁻²) during a 3-week experimental period. Living freshwater pearl mussels were collected from Hemgravs stream in Sweden and transported to Denmark according to permissions granted by the Swedish and Danish authorities.
4. All water samples from stream channels containing empty shells were negative for eDNA indicating that eDNA traces in stream water are most likely to originate from living individuals located upstream of the sampling site. Water samples collected from stream channels containing living individuals of M. margaritifera were consistently positive for eDNA except for one sample (interpreted as a false negative).
5. The study shows that positive eDNA signals for freshwater pearl mussels most likely reflect the presence of living individuals. Consequently, we suggest that eDNA should be used to locate remaining population fragments of M. margaritifera in deep and turbulent streams, providing a platform for faster and more efficient decision making when launching investigative and mitigation initiatives.

中文翻译：

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死或活 — 在针对淡水珍珠贻贝 (Margaritifera margaritifera L.) 的环境 DNA 调查中，旧的空壳不会提示假阳性结果

1. 来自水样的环境 DNA (eDNA) 越来越多地用于检测水生生态系统中物种的存在和分布。然而，在监测计划中实施 eDNA 之前，各种特定物种的采样或分析问题仍有待解决，以尽量减少假阳性和假阴性结果的频率。例如，淡水珍珠贻贝 ( Margaritifera margaritifera ) 的空壳含有可提取的 DNA（从磨碎的贝壳中化学提取），这表明在种群已灭绝但空壳材料仍然存在的溪流地点存在假阳性样本的风险。
2. 本研究的目的是调查来自M. margaritifera 的空壳和自然降解壳是否会导致水样中的假阳性 eDNA 信号。
3. 在为期 3 周的实验期间，从带有活淡水珍珠贻贝或空壳材料（密度 ~10 个体 m ^-2）的室外河道（位于丹麦莱明）收集水样。活的淡水珍珠贻贝是从瑞典的 Hemgravs 溪流中采集的，并根据瑞典和丹麦当局的许可运往丹麦。
4. 来自包含空壳的河流通道的所有水样的 eDNA 均为阴性，表明河流水中的 eDNA 痕迹最有可能来自位于采样点上游的活体个体。从含有活体M. margaritifera 的河道收集的水样，除了一个样本（解释为假阴性）外，eDNA 一直呈阳性。
5. 研究表明，淡水珍珠贻贝的阳性 eDNA 信号很可能反映了活体个体的存在。因此，我们建议应使用 eDNA在深水湍流中定位玛格丽特氏菌的剩余种群片段，从而在启动调查和缓解举措时为更快、更有效的决策提供平台。