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/).