Limitations of FishCARD
The FishCARD database did not include barcodes for all California Current marine fishes due to a combination of limited resources, difficulties amplifying vouchered tissue samples, and a lack of some vouchered reference material within the Marine Vertebrates Collection of the Scripps Institution of Oceanography. Of the 149 (17.2%) California Current fishes absent from FishCARD, 44.0% (n=66) are rare in the California Current, 14.7% (n=22) are common but not coastal species, and 2.0% (n=3) were introduced estuarine species; only, 18.0% (n=27) were common coastal species (Supplemental Table 7). As such, FishCARD provides coverage for the vast majority of California Current marine fishes, making it an important tool for metabarcoding studies, despite these missing taxa.
The one major shortcoming of FishCARD is that 20.8% (n=31) of the missing taxa are in the Genus Sebastes, and rockfish are ecologically important (Hyde & Vetter, 2007), form the basis of many commercial and recreational fisheries (Lea, McAllister, & VenTresca, 1999; Williams, Levin, & Palsson, 2010), and declines in rockfish stocks led to the establishment of the largest marine protected areas in southern California, the Cowcod Conservation Areas (Thompson et al., 2017). Unfortunately, this shortcoming cannot be easily overcome through additional 12S barcoding. This is because rockfish are a recent and diverse radiation comprised of 110 species (Ingram & Kai, 2014) and12S fails to resolve most Sebastes to species-level (Hyde & Vetter, 2007; Yamamoto et al., 2017). Thus effective metabarcoding ofSebastes will require designing novel Sebastes- specific metabarcoding primers that target a more rapidly evolving region of the mitochondrial genome (e.g. CytB ) (Thompson et al., 2017).
However, FishCARD includes 100% of all non-Sebastes nearshore species monitored by the Channel Islands National Kelp Forest Monitoring Program (n=80, Sprague et al., 2013), as well as by PISCO, the Partnership for Interdisciplinary Studies of Coastal Oceans (n=76; (Caselle, Rassweiler, Hamilton, & Warner, 2015; Pondella et al., 2015). Further, there is now 12S reference sequence for 98 of the 100 most abundant ichthyoplankton species collected by the California Cooperative Oceanic Fisheries Investigation (CalCOFI) from the California Current between 1951-2019 (only Showy bristlemouthCyclothone signata and Spotted barracudina Arctozenus risso ) (Moser, 1993). Moreover, in real world application, this reference barcode database assigned taxonomy to over 90% of vertebrate ASVs detecting a broad range of ecologically and commercially important nearshore rocky reef species (Pondella II et al., 2019). As such, FishCARD represents an important genetic resource for coastal California marine metabarcoding monitoring efforts.