COI Barcoding

All samples used in this study were sequenced following protocols established by the Consortium for the Barcode of Life (Ivanova et al., 2005, 2006) and protocols and primers used in Cardoso et al. (2015b). Total genomic DNA was isolated from muscle tissue using DNeasy Tissue Kit (Qiagen), following the manufacturer’s instructions. A portion (661 bp) of the 5’-end of the mitochondrial CO1 gene was amplified by polymerase chain reaction (PCR) using the primers LIICO1F (GATTTTTCTCAACTAACCAYAAAGA) and LIICO1R (ACTTCTGGGTGTCCGAARAAYCARAA). PCR mixes included 6.25 μL of 10% trehalose, 2 μL ultrapure water, 1.25 μL of 10× PCR buffer, 0.625 μL MgCl₂ (50 mM), 0.125 μL of each primer (0.01 mM), 0.0625 μL of each dNTP (0.05 mM), 0.0625 μL Taq polymerase, and 2.0 μL DNA template. PCR was carried out on a Veriti 96-Well Thermal Cycler (Applied Biosystems, Inc.), under the following conditions: 3 min at 94°C; 40 cycles of 25 s at 94°C, 40 s at 52°C, and 45 s at 72°C; and 5 min at 72°C. Amplified products were checked on 1% agarose gels. PCR products were labeled with BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (ABI) using standard methods and were bidirectionally sequenced on an ABI 3500 DNA Analyzer capillary sequencer following the manufacturer’s instructions. Alignment was made in Geneious R9¹ (Kearse et al., 2012), mapping new sequences to existing sequences from GenBank and using the consensus of both forward and reverse sequences. All sequences had HQ scores above 88%, no gaps or ambiguous sites were included and no stop codons found. These sequences were submitted to the Barcode of Life Database² under the project “Cytogenetics and Barcoding of Gymnotiformes” (Samples BCG00104–BCG00140).