False positives and negatives
Long-term fish surveys of the studied reservoirs provided robust
background data on the expected distribution and abundance of individual
species that allows these reservoirs to serve as models to study the
mechanisms underlying spatial fish distribution. Gillnets are most
commonly used to sample fish despite their invasiveness, size- and
species-selectivity (Blabolil et al., 2016). Plentiful long-term
(Znachor et al., 2020) and detailed spatially-resolved seasonal data
(Rychtecký & Znachor, 2011) as well as gillnet data dating back to 2004
(Blabolil, et al., 2017b; Říha et al., 2015) are available for Římov.
Klíčava was intensively studied for decades after dam building
(Straškrabová & Pivnička, 2001), and together with Žlutice, is studied
for current ichthyological research interests (e.g. Vejřík et al.,
2019). Three and five gillnet campaigns in Klíčava and Žlutice
respectively are supported by data from other sampling methods (i.e.
electrofishing, fyke-nets, seining, hook lines and trawling) as well as
expert knowledge about fish-habitat relationships (Blabolil et al.,
2017b) All of these reservoirs are also routinely monitored by the
Vltava River authorities for water chemistry and biological parameters
(www.pvl.cz).
Based on expert knowledge and long-term monitoring of these reservoirs
and their fish fauna, only a single taxon was identified as a clear
false positive. Capelin, a marine fish of the North Atlantic, is likely
environmental contamination, possibly through the use of Northern pike
bait. No target fish DNA was found in the PCR positive and negative
controls or the field/filtration, evaporation, and extraction blanks,
which indicates that the field and laboratory protocols used were
effective in avoiding contamination. Only one species, burbot, was
previously observed in Římov (last caught in spring 2011) and not
detected by eDNA metabarcoding. Burbot is the only member of the family
Gadidae in Czech waters, therefore misclassification is highly
improbable. However, rarer eDNA templates are more likely to be missed
during water sampling or lost during molecular workflow processing
(Kelly et al., 2019). Burbot is active in cold water, thus eDNA sampling
in winter or early spring may improve detection (Blabolil et al., 2018).
We performed three PCR replicates per sample in order to reduce
stochastic variation, but greater replication may have increased
detection probability of rare species within individual samples (Harper
et al., 2019b).
Some fishes, such as asp and rudd or European perch and pikeperch,
belong to different genera, but sequences amplified using the 12S-V5
primers were identical. These species pairs were excluded from
downstream analyses because of assignment uncertainty and differing
ecologies, i.e. piscivorous asp and pikeperch preferring open water, and
invertivorous European perch and omnivorous rudd preferring structured
habitats (Blabolil et al., 2016, 2017a, 2017b). Taxonomic coverage,
resolution, and accuracy could be improved for these and other
genetically similar taxa (e.g. sturgeons and coregonids) by using
different or additional primers that target bony fish, such as MiFish
(Miya et al., 2015) and Teleo (Valentini et al., 2016). For taxonomic
assignment, we used BLAST at 99% identity against a reference database
for UK fishes (Hänfling et al., 2016) that was supplemented with
sequences for missing Czech fishes. Reference databases should ideally
comprise sequences from specimens occurring in the study area for all
species to account for potential phylogeographic variation as species
can be locally adapted (Delrieu-Trottin et al., 2019). With a more
comprehensive reference database, it may be possible to use 100% BLAST
identity for taxonomic assignment.