Atlantic salmon (S. salar) coding mRNA sequences (12,062 sequences)54 (link) were translated into protein sequences. Blastp reciprocal best hits between these salmon and trout proteins were aligned with MUSCLE55 (link)56 (link), and rates of silent substitution (dS values) of the corresponding coding sequences were calculated using the Yang and Nielsen method in PAML4.4 (ref. 57 (link)). Ohnologous gene sequences from fish genomes were obtained from Ensembl Treebest gene trees and DCS analyses. MUSCLE alignment of protein sequences followed by PAML4 analysis of the coding sequences (CDS) was used to compute the dS and dN values for pairs of ohnologous sequences originating from the Ts3R WGD in stickleback, tetraodon, medaka and zebrafish, and for trout Ss4R ohnologues. Trout Ts3R ohnologues represent a special case, because each copy (for example, A and B) was further duplicated by the Ss4R, and thus may be represented by one (if the other Ss4R ohnologue was lost) or two sequences (for example, A1, A2 and B1, B2). A given Ss4R ohnologue was always aligned separately to each Ss4R duplicate copy stemming from the Ts3R (for example, A1 to B1 and B2), when they existed. Alignments were then concatenated to compute dS values, which thus represents an average dS (resp. dN) value for the Ts3R duplication for a given family of ohnologues. When Ss4R ohnologues existed in more than two copies, because of subsequent local duplication (for example, A1, A2, A3), we aligned each possible combination of pairs using MUSCLE55 (link)56 (link) (for example, A1–B1, A2–B1, A3–B1, etc.) and then concatenated alignments as before (for example, A1–B1 with A2–B1), in all possible combinations of two concatenated alignments, each leading to a dS (resp. dN) value. The smallest dS value among all alignments was considered the most conservative and retained for further analysis, together with the corresponding dN. The rate of selective constraints on orthologues and ohnologues was calculated with PAML4.4 (ω=dN/dS) using the method of Yang and Nielsen58 (link). A linear extrapolation from the dS comparison was used to infer the timing of the Ss4R.