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Table S1. Specimens used in this study; accession numbers of sequences and collection information. Details of the COI haplotypes are also given for the specimens of Neotrygon annotata, N. kuhlii, and N. ningalooensis.

Table S2. Kimura 2-Parameter (K2P) corrected Neighbor-Joining distance matrix with minimum and maximum values (in brackets) for the Neotrygon kuhlii mitochondrial COI clades 1 to 9.

Table S3. K2P-corrected neighbor-joining distance matrix of Neotrygon species calculated from the COI alignment. Genetic distance is along the lower diagonal and standard error along the upper diagonal.

Table S4. Mean divergence times and their 95% HPD confidence intervals based on fossil calibrations and published COI mutation rates (0.58%/My and 0.9%/My) calculated in the spotted eagle ray (Aetobatus narinari) from the isthmus of panama closure for nodes 1 to 10.

Figure S1. Substitution frequencies of transitions (cross) and transversions (triangle) for: a) the 1st, b) 2nd, and c) 3rd codon positions of the partial COI gene. Observed frequencies are calculated with the K2P genetic distance.

Figure S2. Substitution frequencies of transitions (cross) and transversions (triangle) for: a) the 1st, b) 2nd, and c) 3rd codon positions of the partial 16s gene. Observed frequencies are calculated with the K2P genetic distance.

Figure S3. Substitution frequencies of transitions (cross) and transversions (triangle) for: a) the 1st, b) 2nd, and c) 3rd codon positions of the partial RAG1 gene. Observed frequencies are calculated with the K2P genetic distance.

Figure S4. Mismatch distributions for pairwise comparisons of Neotrygon kuhlii COI mitochondrial clades. Monomorphic clades 3, 4, and 6 were excluded from the calculation. The frequencies of expected and observed differences are given as a solid and dashed line, respectively.

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