The between-seasons nucleotide distance means were computed as the arithmetic mean of all pair wise distances between two seasons in the inter-season comparisons using the MEGA v.3.1 software [68] . The global rate between dN and dS substitutions and the individual sitespecific selection pressure were measured by the likelihood based single likelihood ancestor counting (SLAC) method in Datamonkey (modified Suzuki-Gojobori method) [70, 71] . For datasets over 100 sequences (H3 n = 204 and N2 n = 166) the HyPhy package [72] was applied. The estimations are likelihood-based, employing a codon model cross between HKY85 and MG94. To elucidate single, positively selected amino acids, the HA and NA datasets were analysed with SLAC and a two rate fixed effects likelihood (FEL) [73] using a likelihood approach with neighbor joining phylogenetic trees (HyPhy). Sites with dN>dS with a <0.05 significance level for likelihood ratio test (LRT) were implied as positively selected for the large HA and NA datasets. For the small datasets (genes coding for the internal proteins <15 and the H1N1 viruses <30) we additionally ran a random effects likelihood (REL) analysis using an empirical Bayes approach with NJ phylogenetic trees in Datamonkey [70] . This method is expected to calculate positively selected sites more accurately in small datasets. The accepted significance level for a positively selected site was set at <0.1 (two-tailed binominal distribution) for SLAC and FEL analyses and >50 bayes factor for REL.