2.3. Data analysis

The DNA sequences of the fillets and samples from the reference database were edited and aligned using Bioedit 7.0.9.0 (Hall, 1999), and the haplotypes identified were used in the subsequent analyses.

The fillet sequences were compared to those of the reference database and then with the public databases of the Barcoding of Life Database (BOLD: http://www.barcodinglife.org) and GenBank (http://www.ncbi.nlm.nih.gov), using the BLASTn search tool (Altschul et al., 1990). In all cases, the criterion of identification at species level was that adopted by BOLD, which considers that individuals of the same species have a maximum genetic divergence of 2%. Each species identified was attributed the scientific and common names defined in the decree NI 29/2015, and where more than one common name is assigned to a species, we adopted only the name(s) used in the Northern region of Brazil.

The haplotypes of the fillets, reference species, BOLD and GenBank sequences were aligned in Bioedit and the Fasta file was used in MEGA 10 (Kumar et al., 2018) to calculate the within- and between-taxon genetic divergence, based on the uncorrected p distances, and to generate the Maximum Likelihood cladogram using GTR + I + G model of nucleotide substitution selected by jModeltest 2.1.10 (Darriba et al., 2012; Guindon and Gascuel, 2003). The cladogram was generated to represent the clusters of the species identified in the fillets, using Chanus chanus and Gonorynchus abbreviatus as outgroups, members of the Order Gonorynchiformes, which was considered the common evolutionary ancestor of the taxa identified in this study in the phylogenetic review of bony fish (Betancur-R et al., 2017). The statistical support of the branches was evaluated through 1,000 bootstrap pseudoreplicates. The tree was visualized and edited in FigTree 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).

Subsequently, the relative frequency of the species identified and the total substitution rates of the fillets of each of the study species were plotted in MS Excel 2013. The substitution rates were calculated for the batches packaged by both the supplier companies and the supermarkets. Then, the Chi-square test was performed in MS Excel 2013, to assess whether there were significant differences between the product's origin and the replacement rates.