Abstract
Many species of amphibians in Northern Europe are threatened and the local distributions are rarely described in detail. Application of modern molecular methods provides an important supplementary tool for monitoring the distribution and diversity of amphibians. For this purpose, we designed, tested, validated, and optimized 14 species-specific assays on genomic DNA extracted from tissue samples to use for quantitative polymerase chain reaction (qPCR) setups targeting mitochondrial DNA from amphibians in freshwater samples. The tests confirmed species specificity for all assays. Considering a systematic definition of the limit of detection for each of the assays, the presented qPCR assays are unlikely to return false positive detection from any co-occurring species in northern Europe. For field validation, the qPCR assays were applied in a large-scale nationwide citizen science project in which sampling and qPCR analysis was carried out by high school students. Data from the citizen science project returned the expected results when compared to the known regional distribution of the target species and confirmed the presence of nine out of 14 Danish species of amphibians in the collected freshwater samples. Four out of 2550 qPCR test sets carried out by the high school students required a professional reanalysis in multiple replicates due to initial unexpected results. This emphasizes that efforts from citizen science may generate large amounts of valuable data, as long as the results are carefully scrutinized by experts.
Originalsprog | Engelsk |
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Tidsskrift | Environmental DNA |
Vol/bind | 5 |
Udgave nummer | 6 |
Sider (fra-til) | 1429-1448 |
Antal sider | 20 |
ISSN | 2637-4943 |
DOI | |
Status | Udgivet - 2023 |
Bibliografisk note
Funding Information:This study was supported by the Innovation Fund Denmark (Grant J.nr. 104‐2012‐1), and by the Danish Environmental Agency [Miljøstyrelsen], and the Lundbeck Foundation. We are grateful for the help we received from all the high school students who attended the “DNA & Liv” course and contributed with samples and help on laboratorial analysis. We also thank Pernille Selmer Olsen from the former GeoGenetic laboratory at the Natural History Museum of Denmark (NHMD) for help with the order of reagents and consumables, and Pernille Hjort at NHMD for initial coordination of the “DNA & Liv” course. We also thank all other employees at the Natural History Museum of Denmark who over the past six years occasionally have helped out getting the “DNA & Liv” course running.
Publisher Copyright:
© 2023 The Authors. Environmental DNA published by John Wiley & Sons Ltd.