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.