Aarhus University Seal / Aarhus Universitets segl

No 224: Investigation of occurrence of river pearl oyster (Margaritifera margaritifera) in Varde Å using eDNA

Andersen, L.W. & Wiberg-Larsen, P. 2017. Undersøgelse af forekomsten af flodperlemusling (Margaritifera margaritifera) i Varde Å ved brug af eDNA. Aarhus Universitet, DCE – Nationalt Center for Miljø og Energi, 28 s. - Videnskabelig rapport fra DCE - Nationalt Center for Miljø og Energi nr. 224. http://dce2.au.dk/pub/SR224.pdf



Very little is known about the status of the River Pearl Mussel, Margaritifera margaritifera, at its only historically (and recently) known Danish habitats in River Varde in Western Jutland, and it has been doubted if the species still holds populations there. The species is globally threatened and protected according to the EU Habitats Directive.

Using environmental DNA (eDNA) this report documents that the mussel occurs in River Varde, being located upstream “Vagtborghus” and further downstream at Varde City. It was, however, not found at Sig located 6 km upstream “Vagtborghus” outside its known distributional area.

The presence of the species was also investigated at two streams in Blekinge (South Sweden) where it occurs in high and low density, respectively, and three additional Danish streams (in South Zealand and Funen) where it was not expected to occur. We analysed for eDNA in both 15 mL water samples and samples of the upper 1.5 cm of sandy sediment. Triplicate samples were taken at all sites, and each of these analysed by three subsamples.

All extractions of eDNA were carried out using PowerSoil kit (MoBio), thereby minimising unwanted soluble organic compounds and particles in the solvents. The extracted eDNA was subsequently analysed using the primer MmaCOI, optimised with the qPRC-enzyme KAPA SYBR® FAST (KEM-EN-TECH) to be specific for M. margaritifera before carrying out qPCR to amplify present eDNA segments. The “limit of detection” (assessed as Ct) was estimated from a number of dilutions of a known DNA concentration using visual inspection of the resulting amplification-plots. To decide if the mussel was present in a given sample, at least two out of the three triplicate plots should be positive and further the obtained values should be within the Ct-range. Further, positive amplification-plots were additionally run on gel to verify if the plots represented a PCR-band of the expected size for the species. Similar analyses were run on negative controls (i.e. additional samples without including eDNA).

The presence of M. margaritifera was confirmed from water as well as sediment samples at two sites in River Varde, the two South Swedish streams, but not (as expected) the other three Danish stream sites (except for one obviously “false positive” sample in one of these) and the most upstream site in River Varde.

The results are not sufficient to establish the precise distribution of the population in River Varde as excreted eDNA may be transported (and dispersed) and, thus, still being detectable over several kilometres. However, because general dilution of eDNA may be extensive due to the high discharge, there are reasons to believe that the population may be of significant size (based on comparison of discharge and population size in the two Swedish streams and the discharge in River Varde). Unfortunately, no conclusions can be made about age structure and recruitment of the population. Thus, despite the obvious potential of eDNA analyses, supplementary traditional monitoring (visual inspection and sampling of specimens) is needed to assess the conservation status of the species in Denmark and to conclude whether the population is viable.

Assuming that the population in River Varde has growth rates similar to those reported for rivers in Mid-Sweden, a single specimen (length 102 mm) caught coincidently in the autumn 2000 may have been 50-60 years old and thus potentially fertile.  If so, it cannot be excluded that the population still have the ability to reproduce.