Corresponding authors: Jan Martini (
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The rare mayfly
We developed and validated a targeted qPCR protocol to detect this mayfly and applied it to standardized water samples filtered for eDNA analysis from the Vjosë (Albania) and Volga (Russia), two rivers with extant populations of
At each sampling site in the Vjosë, eDNA samples were collected by filtering 0.5 L of stream water through each of two 0.45 µm Sterivex filters. In the Volga, 2 L of stream water were filtered through a total of eight 0.7 µm glass fibre filters. Filters were stored and shipped at -20°C until further processing. Environmental DNA extraction was performed using the DNeasy® PowerWater® Sterivex™ Kit following the Experienced User protocol for Vjosë samples and via a Phenol-Chloroform Isoamyl extraction for the Volga samples. A Taqman qPCR assay was developed using a newly designed primer and probe set. Standard curves obtained from an amplicon dilution series yielded a reaction efficiency of 88% and an R2-value of 0.995, with a calculated limit of quantification of 1801 copies/µL and a limit of detection of 59 copies/µL. Each eDNA sampling replicate was tested using five qPCR replicates, yielding ten qPCR reactions per sampling site in the Vjosë river and 40 qPCR reactions in the Volga river. With each batch of eDNA samples, four negative and two positive controls were analysed.
At each site, we also collected benthic MHS samples with a 25x25 cm 500 µm net where 20 samples were taken to reflect microhabitat distribution and dominance. Benthos samples were subsampled in the lab and all zoobenthos hand-picked.
While
We demonstrate the principal applicability of molecular methods to search for rare species in hot-spots of biodiversity in Central Europe. The remnant populations of
Jan Martini
1st DNAQUA International Conference (March 9-11, 2021)
ERC-Starting Grant #716196 to Gabriel Singer.
Martin Schletterer acknowledges support of two organizations: Sampling at the Volga river took place within the framework of the LTERM project REFCOND_VOLGA; the eDNA survey in 2017 was funded by VÖU.
ERC-Starting Grant #716196 to Gabriel Singer.