Extending sampling approaches for great crested newt ( Triturus cristatus ) eDNA monitoring

Abstract Environmental DNA (eDNA) monitoring has been used for great crested newt ( Triturus cristatus ) survey in the UK since the publication of a Defra-funded trial in 2014. If eDNA results are to be used in support of a great crested newt licence, surveys must be performed during a 76-day survey window (15 April – 30 June) to coincide with peak great crested newt activity, and must follow the approved ethanol precipitation protocol. However, eDNA detection is possible in other months and filtration may be a more effective method of eDNA capture. We investigated whether the great crested newt eDNA survey season could be extended and filtration could be used for great crested newt eDNA capture by reviewing the available evidence and conducting a field study from April to October 2022. Paired water samples for ethanol precipitation and filtration were collected from 25 ponds once a month, resulting in 124 samples of each type. All samples (N = 248) were analysed with the approved great crested newt quantitative PCR assay. Our results indicate that great crested newts can be reliably detected using both eDNA capture methods from April to August, with detection rates decreasing in September and October. Great crested newt eDNA detection was comparable or higher with filtration than ethanol precipitation. Practical implication. Acceptance of filtration for great crested newt eDNA surveys could allow more water to be processed for robust and reliable estimates of great crested newt presence. Extending the great crested newt eDNA survey season to August could allow more waterbodies to be surveyed for great crested newt presence (but not absence), and identification of sites that provide important habitat for great crested newts outside of the breeding season. This would also remove logistical challenges and costs associated with completing sampling within 11 weeks and laboratory analysis within 10 working days from sample receipt. Furthermore, great crested newt eDNA surveys could be more frequently carried out alongside monitoring for other species, which are typically surveyed from April to September/October or year-round with conventional methods or eDNA surveys using filtration. This could enable infrastructure projects to develop more effective mitigation measures as well as reduce time required from surveyors and survey costs.