Phylogenetic Network and Recombination Analyses

A recent study found evidence for intragenic recombination within the PVA genome (Fuentes et al., 2021). To investigate the role of recombination, we used two different methods to investigate the occurrence of recombination events in CP sequences. A phylogenetic network was first reconstructed using the neighbor-net method implemented in SplitsTree v4.14.8 (Huson, 1998) with default settings. The pairwise homoplasty index (PHI) test implemented in SplitsTree was also carried out to test signals of recombination (p < 0.05, significant evidence of recombination). In addition, to confirm the occurrence of recombination in our dataset, localization of recombination breakpoints and identification of likely parental sequences were achieved with the RDP v4.101 package, which incorporates the algorithms RDP, Geneconv, Bootscan, Maxchi, Chimaera, Siscan, and 3Seq (Martin et al., 2015). Recombination events supported by at least four different algorithms of analysis and with p values < 1.0 × 10^–5, viral isolates were identified as recombinants. Because recombinants may result in misleading results in selection analysis, as reported by Anisimova et al. (2003) and Sironi et al. (2015), the recombinants were excluded from subsequent selection analysis.