Natural England Commissioned Report NECR286 Developing DNA applications within Natural England for freshwater mussels, saline lagoon species and terrestrial invertebrates in 2017/18 First published 21 May 2020 www.gov.uk/natural-england Foreword Natural England commission a range of reports from external contractors to provide evidence and advice to assist us in delivering our duties. The views in this report are those of the authors and do not necessarily represent those of Natural England. Background DNA based applications have the potential to significantly change how we monitor biodiversity and which species and taxa we monitor. These techniques may provide cheaper alternatives to existing species monitoring, an ability to detect species that we do not currently monitor effectively and the potential to develop new measures of habitat and ecosystem quality. Natural England has been supporting the development of DNA techniques for a number of years. The use of environmental DNA (eDNA) to determine the presence or absence of great crested newts in ponds is now a standard tool for developers and consultants. There are still significant limitations to the use of this technology in other areas and in 2017/18 Natural England worked with NatureMetrics to prove the concept of using DNA for monitoring terrestrial invertebrates, saline lagoon species and freshwater mussels. This report presents the results from those proof of concept studies, which were promising, and suggests areas where further development could be targeted. This report should be cited as: TANG, C.Q., CRAMPTON-PLATT, A., & SMITH, T. 2020. Developing DNA applications within Natural England for freshwater mussels, saline lagoon species and terrestrial invertebrates in 2017/18. Natural England Commissioned Reports, Number 286 Natural England Project Managers– Gavin Measures, Maija Marsh, Jon Webb and Andy Nisbet Contractor - NatureMetrics Keywords – DNA, eDNA, terrestrial invertebrates, metabarcoding, saline lagoon invertebrates, freshwater mussels, Further information This report can be downloaded from the Natural England Access to Evidence Catalogue: http://publications.naturalengland.org.uk/ . For information on Natural England publications contact the Natural England Enquiry Service on 0300 060 3900 or e-mail [email protected]. This report is published by Natural England under the Open Government Licence - OGLv3.0 for public sector information. You are encouraged to use, and reuse, information subject to certain conditions. For details of the licence visit Copyright. Natural England photographs are only available for non-commercial purposes. If any other information such as maps or data cannot be used commercially this will be made clear within the report. ISBN 978-1-78354-580-3 © Natural England and other parties 2020 Executive Summary ................................................................................................................ 2 Developing eDNA sampling methods to monitor freshwater mussel populations in England ..................................................................................................................................... 3 Introduction ........................................................................................................................... 3 Methods ................................................................................................................................ 4 Results .................................................................................................................................. 8 Discussion .............................................................................................................................. 12 Annex 1 ............................................................................................................................... 14 Annex 2 ............................................................................................................................... 16 Lagoon invertebrate eDNA .................................................................................................. 17 Introduction ......................................................................................................................... 17 Nematostella vectensis eDNA assay ................................................................................. 19 Methods .............................................................................................................................. 19 Results ................................................................................................................................ 22 Discussion .......................................................................................................................... 27 Future developments ............................................................................................................ 30 Nematostella vectensis eDNA assay ............................................................................. 30 Gammarus insensibilis eDNA assay .............................................................................. 31 Metabarcoding of invertebrate samples from vane traps and from riparian pitfall traps .................................................................................................................................................. 32 Scope .................................................................................................................................. 32 Methods .............................................................................................................................. 32 Results ................................................................................................................................ 34 Comparison with morphological data ............................................................................. 41 Discussion .......................................................................................................................... 42 Conclusion .......................................................................................................................... 44 Appendices are available as separate downloads ....................................................... 45 Appendix 1 ......................................................................................................................... 45 Appendix 2. ........................................................................................................................ 45 Appendix 3. ........................................................................................................................ 45 Appendix 4. ........................................................................................................................ 45 Appendix 5. ........................................................................................................................ 45 Appendix 6. ........................................................................................................................ 45 Appendix 7. ........................................................................................................................ 45 Appendix 8 ......................................................................................................................... 45 1 NatureMetrics Ltd, CABI site, Bakeham Lane, Egham, Surrey, TW20 9TY Executive Summary In 2017/18, Natural England commissioned NatureMetrics to conduct proof of concept studies using DNA for: 1. Developing eDNA sampling methods to monitor freshwater mussel populations in England The UK is home to 6 species of freshwater Unionoida mussels, each with a different distribution. Of these 6 species, the depressed river mussel (Pseudanodonta complanata) and the pearl mussel (Margaritifera margaritifera), have experienced significant declines during the 20th century, and are now protected species. Traditional survey methods however are expensive, time consuming, and have a significant risk of missing their presence at low densities. Environmental DNA (eDNA) methods may offer a viable alternative solution that is quick and sensitive. A set of primers that specifically amplify British Unionid mussels were developed in this study which can be used detect the 6 native freshwater Unionid mussels in the UK. When the sample is likely to contain only a single species, the resulting amplicons can be Sanger sequenced to provide a quick and reliable species determination, however the assay requires further validation. 2. Lagoon invertebrate eDNA The starlet sea anemone, Nematostella vectensis, and the lagoon sand shrimp, Gammarus insensibilis, are saline lagoon specialist protected species. However, like other lagoon specialist species, monitoring these species using traditional survey techniques can be notoriously difficult, due to their small size and cryptic nature. In this study a set of primers were developed that specifically amplified Nematostella vectensis. These primers appear specific to N. vectensis but require more validation. Specific primers to use for a Gammarus insensibilis eDNA assay were failed to be developed, due to the paucity of genetic reference data available for this species. Obtaining the first reference sequences relies on the liberal use of universal primers and a great deal of sequencing effort. More research is required to start the development of this assay. 3. Metabarcoding of invertebrate samples from vane traps