Method

1 Genome

A total of 32 genomic sequences of ranaviruses from various cold-blooded vertebrates (reptiles, amphibians and fishes) were obtained from NCBI (National Center of Biotechnology Information). The detailed information about host species, the country of origin and the year of detection are listed in Table S1.

2 Core-pan analysis

In order to identify the core genes of ranavirus, the pan-genome analysis of orthologous gene clusters were carried out using PanX(Ding et al., 2017) for the 32 completed ranavirus genomes. The strictly core genes (including genes present in all viral genomes) were identified using PanX(Ding et al., 2017) with the parameters as “-dmi 0.7” and “-nsl”. “-dmi 0.7” means that the sequence identity threshold to report an alignment is 0.7, and “-nsl” means to “disable long branch splitting”.

3 Phylogenetic analysis of genome

Composition vector phylogenetic tree (CV-Tree)(Gao & Qi, 2007; Zuo & Hao, 2015): the amino acid sequences in FASTA format were directly summited in CVTree3 Web Server (http://tlife.fudan.edu.cn/cvtree/cvtree/) and K-tuple length was set at 6. Additionally, the genomic sequences of other Iridovirus family were included as an out-group. Visualization and annotation for phylogenetic tree were performed using Evolview v3 (Subramanian et al., 2019).
Neighbor-joining phylogenetic tree (NJ-Tree) based on ranavirus core genes: the ranavirus core genes identified by core-pan analysis were aligned using MAFFT software (Katoh et al., 2009), and the aligned sequences were concatenated in order using PhyloSuite (Zhang et al., 2018). Finally, the streamlined sequences were used to construct Neighbor-joining phylogenetic tree by using MEGA 6.0 with 1,000 bootstrap replicates(Tamura et al., 2013). NJ-Tree based on iridovirid core genes: iridovirid core genes were selected from the ranavirus core genes identified by core-pan analysis. The subsequent steps were same as Neighbor-joining phylogenetic tree (NJ-Tree) based on ranavirus core genes.

4 Dot plot analysis

Dot plot analysis of ranaviruses was performed using Java Dot Plot Alignments (JDotter) (Brodie et al., 2004). FV3 (NCBI access number is AY548484) was placed in horizontal sequence, meanwhile the other ranaviruses were placed in vertical sequence. Maximum plot size was set at 700 bases and sliding window size was set at 50. The nucleic acid sequences of FASTA format used for Dot plot analysis were obtained from NCBI.

5 Recombination analysis

The ranavirus core genes identified by core-pan analysis were concatenated using PhyloSuite (Zhang et al., 2018). The streamlined sequences were imported into the Recombination Detection Program (RDP) BETA4.67 (Darren P Martin et al., 2015). Putative recombination breakpoints were visualized in RDP 4 software by algorithms of RDP, GENECONV, Bootscan, Maxchi, Chimaera, SiSscan, PhylPro, LARD and 3Seq. UPGMA (unweighted pair group method using arithmetic average) phylogenetic tree was constructed by TREES module within RDP4.

6 Substitution saturation analysis

The ranavirus core genes were imported separately to DAMBE Version 5.3.19 (Xuhua Xia, 2013). The index of nucleotide substitution saturation was estimated using Xia method (Xuhua Xia et al., 2003).