Importance of Complete Reference Databases
Previous eDNA metabarcoding efforts in the California Current reported
poor species-level identification and frequent taxonomic assignment to
non-native sister taxa (Closek et al., 2019; Kelly, Port, Yamahara, &
Crowder, 2014; Port et al., 2015), results that are likely due to the
lack of adequate reference sequences for these species. For example, an
eDNA metabarcoding study in Southern California (Curd et al., 2019)
assigned multiple 12S ASVs to Girella simplicidens , the
Gulf opaleye, a fish native to the Gulf of California that does not
occur in California coastal waters (Froese & Pauly, 2010; Love &
Passarelli, 2020). This incorrect assignment occurred because there were
no corresponding 12S reference sequences for the local native
Opaleye, G. nigricans . By maximizing the number of reference
barcodes from local species, FishCARD allows the vast majority of reads
to be correctly assigned to ecologically and geographically relevant
species.
Compared to the CRUX database generated from 12S fish
sequences in CRUX-12S , FishCARD improved species-level
assignments, identifying an additional 21.8% of the vertebrate reads in
our eDNA samples. Much of this improvement was due to the inclusion of
reference barcodes for Kelp bass (Paralabrax clathratus ), one of
the most abundant marine species in Southern California kelp forest
ecosystems and an important sport fishery species (Pondella et al.,
2015). By including this species, FishCARD assigned 2 previously
unidentified ASVs to P. clathratus, which accounted for 16.4% of
our total sequence reads.