Reference Barcode Generation
To generate a more complete 12S barcode reference database for fish found in the California Current Large Marine Ecosystem, we assembled a list of native marine teleost and elasmobranchs, comprising a total of 864 species (Allen & Horn, 2006; Froese & Pauly, 2010; Hastings & Burton, 2008; Love, & Passarelli, 2020) (Supplemental Table 1). From this list, we acquired as many ethanol-preserved specimens as possible from the Scripps Institution of Oceanography Marine Vertebrates Collection at University of California San Diego (SIO). We obtained a total of 757 samples, representing 612 species (Supplemental Table 2) or 70.8% of all described species of California Current marine fishes. Of these 757 samples, 258 had no previous 12S barcodes.
For each sample, we extracted DNA from ~0.25 mg of tissue in 300 µL of a 10% Chelex slurry (Walsh, Metzger, & Higuchi, 1991). Given the high volume of samples to process, we initially froze sample slurries at -20˚C. Subsequently, samples were thawed, vortexed for 10 seconds, and then centrifuged at high speed for 15 seconds prior to incubating at 95˚C for 20 minutes. Samples were then vortexed and centrifuged again at high speed and stored at 4˚C until use.
We amplified all DNA extracts using the MiFish Universal Teleost Primers and additionally amplified all elasmobranch samples using the MiFish Elasmobranch Primers (Miya et al., 2015). PCR amplification was conducted following the thermocycler profile of Curd et al.(2019b). PCR reactions had 25 μL reaction volume containing 12.5 μL QIAGEN Multiplex Taq PCR 2x Master Mix (Qiagen Inc., Valencia, CA, USA), 6.5 µL of molecular grade water, 2.5 µL of each primer (2 µmol/L), and 1 μL DNA extraction. PCR thermocycling employed a touchdown profile with an initial denaturation at 95°C for 15 min to activate the DNA polymerase, followed by 13 cycles of a 30s denaturation at 94°C, a 30s annealing that started at 69.5°C and then decreased by 1.5°C for each subsequent cycle (last cycle was 50°C), finishing with a 1 min extension at 72°C. This initial touchdown profile was followed by 35 additional cycles using identical parameters except a constant annealing temperature of 50°C and ending with a final extension at 72°C for 10 min. All PCRs included a negative control, where molecular grade water replaced the DNA extraction. All PCR products were visualized via electrophoresis on 2% agarose gels to ensure amplification success and correct product size.
PCR products were purified using ExoSAP-IT (Affymetrix, Cleveland, OH, USA) and sequenced in both directions using BigDye chemistry (Applied Biosystems Inc, Foster City, CA, USA) at Laragen Inc., (Culver City, CA, USA). We trimmed and aligned forward and reverse sequences in Sequencher version 5.4.6 (Nishimura, 2000). All taxonomic names between GenBank and vouchered specimens were synonymized to NCBI taxonomy using the Rpackage taxize (Chamberlain & Szöcs, 2013). The resulting12S sequences were deposited into GenBank (XXXXXX–XXXXXX; Supplemental Table 2).