The BLAST search on NCBI ( and GISAID

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1 Supplemental materials and methods The BLAST search on NCBI ( and GISAID ( showed that hemagglutinin (HA) gene of North American H5N1, H5N2 and H5N8 viruses had >98% nucleotide identities with H5N8 viruses identified from Korea, Japan, and Europe. A total of 70 isolates were selected based on the BLAST results of H5N1, H5N2 and H5N8 viruses identified from North America to construct NJ phylogenetic trees (Supplemental table 1). Neighbor-joining (NJ) phylogenetic trees were constructed for each of the 8 gene segments. The NJ phylogenetic network and time-scaled phylogenetic tree of the HA gene were constructed using 59 HA gene sequences of group A viruses based on the result of NJ phylogenetic analyses. Specifically, sequences were aligned using MUSCLE (1), and manual editing and tree reconstruction were done using MEGA 6 (2). Tree support was determined by bootstrap analysis using 1000 replicates. Bayesian analysis was done using BEAST v1.7 (3). We used the codon-based SRD06 nucleotide substitution model and the constant coalescent prior. A Markov Chain Monte Carlo (MCMC) method to sample trees and evolutionary parameters was run for 2 x 10 7 generations. BEAST output was analyzed with TRACER v1.4 with 10% burn-in. We ran two independent, identical analyses and compared the log files in TRACER v1.4 to determine convergence. All parameter estimates for each run showed ESS values > 500. A maximum clade credibility (MCC) tree was generated for each data set using TreeAnnotator in BEAST. FigTree ( was used for visualization of trees. The hemagglutinin gene segment was used to construct a phylogenetic network using the Median Joining method implemented in NETWORK ver (4). Nucleotide positions of the HA gene for Bayesian analysis and the phylogenetic network were 49 to 1659.

2 References 1. Edgar RC MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic acids research 32: Tamura K, Stecher G, Peterson D, Filipski A, Kumar S MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular biology and evolution 30: Drummond AJ, Rambaut A BEAST: Bayesian evolutionary analysis by sampling trees. BMC evolutionary biology 7: Bandelt HJ, Forster P, Rohl A Median-joining networks for inferring intraspecific phylogenies. Molecular biology and evolution 16:37-48.

3 Supplemental figure legends Supplemental figure 1. Phylogenetic tree of the HA gene. Unrooted phylogenetic tree was generated by the distance-based neighbor-joining algorithm (nucleotide positions: ). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Supplemental figure 2. Time-scaled phylogenetic tree (months on the horizontal axis) of the intercontinential group A (ica) hemagglutinin clade HPAIV. Nucleotide positions of HA gene for Bayesian analysis was 49 to Subgroup ica1, ica2, and ica3 viruses are marked in blue, red, and green, respectively. Supplemental figure 3. Phylogenetic tree of the NA gene. Unrooted phylogenetic tree of N8 gene segment was generated by the distance-based neighbor-joining algorithm (nucleotide positions: ). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Supplemental figure 4. Phylogenetic tree of the PB2 gene. Unrooted phylogenetic tree was generated by the distance-based neighbor-joining algorithm (nucleotide positions: ). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Supplemental figure 5. Phylogenetic tree of the PB1 gene. Unrooted phylogenetic tree was generated by the distance-based neighbor-joining algorithm (nucleotide positions: ). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches.

4 Supplemental figure 6. Phylogenetic tree of the PA gene. Unrooted phylogenetic tree was generated by the distance-based neighbor-joining algorithm (nucleotide positions: ). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Supplemental figure 7. Phylogenetic tree of the NP gene. Unrooted phylogenetic tree was generated by the distance-based neighbor-joining algorithm (nucleotide positions: ). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Supplemental figure 8. Phylogenetic tree of the M gene. Unrooted phylogenetic tree was generated by the distance-based neighbor-joining algorithm (nucleotide positions: ). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Supplemental figure 9. Phylogenetic tree of the NS gene. Unrooted phylogenetic tree was generated by the distance-based neighbor-joining algorithm (nucleotide positions: ). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches.

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