Detection of a New Non-Native Freshwater Species by DNA Metabarcoding of Environmental Samples – First Record of Gammarus Fossarum in the UK
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Aquatic Invasions (2017) Volume 12, Issue 2: 177–189 Open Access DOI: https://doi.org/10.3391/ai.2017.12.2.06 © 2017 The Author(s). Journal compilation © 2017 REABIC Research Article Detection of a new non-native freshwater species by DNA metabarcoding of environmental samples – first record of Gammarus fossarum in the UK Rosetta C. Blackman1,*, Drew Constable2, Christoph Hahn1,3, Andrew M. Sheard4, Jessica Durkota5, Bernd Hänfling1 and Lori Lawson Handley1 1Evolutionary and Environmental Genomics Group, School of Environmental Sciences, University of Hull, Cottingham Road, Hull, HU6 7RX, UK 2Environment Agency, Bromholme Lane, Brampton, Huntingdon, Cambs, PE28 4NE, UK 3Institute of Zoology, University of Graz, Universitaetsplatz 2, A-8010 Graz, Austria 4Independent author, Beverley, UK 5Department of Geography, University College London, Gower Street, London, WC1E 6BT, UK Author e-mails: [email protected] (RCB), [email protected] (DC), [email protected] (CH), [email protected] (AMS), [email protected] (JD), [email protected] (BH), [email protected] (LLH) *Corresponding author Received: 23 September 2016 / Accepted: 20 March 2017 / Published online: 24 April 2017 Handling editor: Michal Grabowski Abstract We report the discovery of a non-native gammarid, Gammarus fossarum (Koch, 1836) (Crustacea, Amphipoda), in UK rivers. Gammarus fossarum is a common freshwater gammarid in many parts of mainland Europe, but was previously considered absent from the UK. Gammarus fossarum was detected in a number of UK rivers following DNA metabarcoding of a mini-barcode region of the COI gene in macroinvertebrate kick samples, and environmental DNA (eDNA) from water and sediment samples. Subsequent morphological analysis and standard DNA barcoding showed that the species could be reliably identified and separated from Gammarus pulex (Linnaeus, 1758), the most dominant and widespread native freshwater gammarid in the UK. Our data demonstrate extensive geographical coverage of G. fossarum in the UK, spanning distant river catchments. At present there is no data to confirm the likely origin of G. fossarum’s introduction. Subsequent re-examination of historic archive material shows the species to have been present in the UK since at least 1964. This study is among the first to demonstrate the potential of eDNA metabarcoding for detection of new non-native species. Key words: environmental DNA, metabarcoding, passive detection, early warning, cryptic species, Gammaridae, non-native Introduction et al. 2011; Constable and Birkby 2016) and introduce novel pathogens to newly colonised areas (Bacela- Amphipods are successful invaders in freshwater Spychalska et al. 2012). ecosystems, with many invasive non-native species Once non-native species are widely established, (INNS) having been observed to adversely impact efforts to reduce their impacts are often problematic, indigenous species within Europe over the last century hence management strategies are strongly focused (Bij de Vaate et al. 2002; Grabowski et al. 2007). on preventing introductions or spread (e.g. the “check, The introduction of non-native amphipods may not clean, dry” campaign in the UK). Early detection is only lead to displacement of native congeners (e.g. key to such strategies, either to improve the success Dick and Platvoet 2000; MacNeil and Platvoet 2005; of eradication programs or to prevent further estab- Kinzler et al. 2009), but may also impact on ecosystem lishment and dispersal (Roy et al. 2014; Dejean et al. structure and functioning (MacNeil et al. 2011; Piscart 2012). For freshwater macroinvertebrates, INNS detection 177 R.C. Blackman et al. methods typically rely on sampling programmes and brate community and eDNA metabarcoding. The morphological identification. However, the standard species was found in several UK rivers following a UK monitoring method for macroinvertebrates, a preliminary non-targeted sampling programme for three minute kick sample, will typically recover 62% macroinvertebrate communities based on metabarcoding of families and 50% of species at a site (Furse et al. of a 313 bp mini-barcode region of the cytochrome c 1981). This can present considerable challenges when oxidase subunit I (COI) gene, and was subsequently dealing with rare or elusive species. Morphological confirmed using a combination of morphological identification can also prove difficult when identifying analysis and standard full-length COI DNA barcoding taxonomically similar or cryptic species, or juvenile (via Sanger sequencing). This study demonstrates life stages, and is highly dependent on the taxonomical the power of eDNA metabarcoding for detection of expertise of the investigator. Emerging molecular non-native species in natural habitats. detection methods may provide significant benefit for detecting non-native species in aquatic environments Methods (Darling and Mahon 2011; Lawson Handley 2015). One new and rapidly developing method is the Metabarcoding surveys use of environmental DNA (eDNA) (Taberlet et al. 2012a, b; Rees et al. 2014; Lawson Handley 2015), Sampling which refers to cellular or extracellular DNA that Field surveys were carried out in March 2015 within can be extracted directly from environmental samples 8 UK river catchments (Figure 1, Maps A–H, excluding without prior separation of taxa (Taberlet et al. E). At each site (n = 65) environmental variables 2012a). Environmental DNA has been successfully including water depth, width, substrate type and used in numerous studies to detect specific taxa surrounding habitat were recorded. Three sample using a targeted approach based on standard or types were collected at each site: a three minute quantitative PCR (Dejean et al. 2012; Dougherty et macroinvertebrate kick sample (Murray-Bligh 1999) al. 2016). In an alternative approach, called “meta- for identification by microscopy analysis and high barcoding”, entire species assemblages are analysed molecular weight DNA extraction from pools of by PCR with broadly conserved primers, followed individuals; and water and sediment samples were by Next Generation Sequencing (NGS: see Lawson collected for eDNA extraction. Two litres of water Handley 2015; Hänfling et al. 2016; Port et al. 2016; was sampled from the surface by collecting 4 × 500 ml Valentini et al. 2016 for further detail). Environ- from points across the river width using a sterile mental DNA metabarcoding has been successfully bottle. Sediment samples were collected from points used in a small number of studies, for example, to across the river width using a trowel, and the describe entire communities of vertebrates (e.g. material was placed in a 42 fluid oz. sterile Whirl-pak® Lawson Handley 2015; Hänfling et al. 2016; Port et bag (Cole-Palmer, Hanwell, London). All sampling al. 2016; Valentini et al. 2016) and invertebrates equipment was sterilized in 10% commercial bleach (Deiner et al. 2016) from marine, lake and river solution for 10 minutes then rinsed with 10% MicroSol samples. Metabarcoding has excellent potential as an detergent (Anachem, UK) and purified water between early warning tool for detection of non-native species samples. Sample bottles filled with ddH2O were from samples collected from invasion pathways or taken into the field and later filtered as sample blanks. natural/semi-natural habitats (Mahon and Jerde 2016; Lawson Handley 2015). For example, the technique Macroinvertebrate community sample processing was recently used as an early detection method for screening ship ballast, and detected non-indigenous All macroinvertebrates from each kick sample were zooplankton in Canadian ports (Brown et al. 2016). sorted and identified to the lowest taxonomic level Environmental DNA metabarcoding has also identified possible, before being stored in sterile 50 ml falcon non-native fish species present in samples from the tubes filled with 100% ethanol. For DNA extraction, live bait trade (white perch, Morone americana samples were dried to remove the ethanol and the (Gmelin, 1789) Mahon et al. 2014) and in river entire macroinvertebrate community was lysed in a samples (northern snakehead, Channa argus (Cantor, Qiagen Tissue Lyser® with Digisol (50mM Tris, 20M 1842) Simmons et al. 2015). However the number of EDTA, 120 mM NaCl and 1% SDS) (3 × 30 sec). applications of metabarcoding for detection of non- Samples were then incubated overnight at 55 °C with native species has so far been limited. SDS and Proteinase K. DNA from a 200 μl subsample In this paper we describe the detection of of the lysed tissue was extracted using the DNeasy Gammarus fossarum (Koch, 1836), a newly recognised Blood & Tissue Kit® (Qiagen, Hilden, Germany) freshwater amphipod to the UK, using macroinverte- according to the manufacturer’s protocol. 178 Detection of a new non-native freshwater species by DNA metabarcoding Figure 1. Distribution of Gammaridae species detected during this study. – Gammarus fossarum, – Gammarus pulex and – both species present. A – River Hull, B – River Bain, C – River Cam, D – River Colne, E – Nailbourne, F – River Frome, G – Rivers Taff and Ely and H – River Ribble. See supplementary information Table S1 for further site information (Pebesma et al. 2005; Wickham. 2009; Bivand et al. 2013; Bivand et al. 2016; Gallic. 2016) Contains OS data © Crown copyright and database right (2016). 179 R.C. Blackman et al. systems Veriti Thermal Cycler with the following Environmental DNA sample processing profile: initial denaturation at 95 °C