2.5. Phylogenetic analyses
Because of its novelty, the taxonomy status of PCV4 has not been determined. Additionally, with the extreme diversity of CRESS DNA viruses (a group of circular, single-stranded DNA viruses encoding replication- associated protein) (Rosario, Duffy, & Breitbart, 2012; L. Zhao, Rosario, Breitbart, & Duffy, 2019), it was our curiosity to have a broad visualization of the genetic relationship of PCV4 with others CRESS DNA viruses. As a result, the phylogenetic analyses were done based on amino acid alignments of replication- associated protein (Rep), utilizing reference sequences described in the previous studies (Kazlauskas, Varsani, & Krupovic, 2018; Krupovic et al., 2020; Rosario et al., 2017). The alignment of the putative Rep sequences was done by COBALT tool (Papadopoulos & Agarwala, 2007). Complete lists of Rep sequences were given in Supplementary data S1- S2.
Using IQ-TREE v2.1.1 (Minh et al., 2020), the genetic relationships between CRESS DNA viruses were inferred by maximum likelihood method. The ‘-m MFP’ option was invoked which helps selecting the data best-fit amino acid substitution model. The branch support values were estimated by ultrafast bootstrap approximation (Hoang, Chernomor, von Haeseler, Minh, & Vinh, 2018) via “-bb 1000” option. All phylogenies were midpoint rooted and displayed by FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).