Assessing the impact of phylogenetic reconstruction error on genetic reassortment inference in segmented viruses.

Recombination is an important process in the evolution of RNA viruses. For viruses with segmented genomes, recombination can occur by reassortment of entire genomic segments. This process has played a significant role in the adaptation and spread of influenza viruses with novel genomic combinations, i.e., antigenic shifts. Reassortment is usually identified by discrepancies between the topologies of trees reconstructed from different genomic segments, e.g., subtree prune and regraft (SPR) rearrangements. However, this phylogenetic discordance can also result from errors in tree reconstruction due to insufficient information. The objective of this study was to evaluate the contribution of phylogenetic reconstruction error to the inference of reassortment events from influenza A virus (IAV) genomes from avian hosts. We curated a dataset of 11,795 complete avian H5Nx IAV genomes and sampled subsets of HA sequences with different expected levels of reconstruction error. Next, we simulated molecular evolution along the trees reconstructed from these HA samples, varying rates of evolution and sequence lengths to correspond to the other genomic segments. Because these simulations had no reassortment, any phylogenetic discordance identified from the resulting trees must be caused by reconstruction error. We found that simulations from random samples yielded about 32% as many SPRs as obtained from real data. This false positive rate was reduced to 4% when simulating trees from the most genetically divergent subset of HA sequences. By modifying the simulated trees with SPR rearrangements, we observed a false negative rate of 8.5%, which increased with the number of SPRs per tree. Overall, we found that reassortment can be substantially overestimated from phylogenetic discordance, underscoring the need to account for phylogenetic uncertainty and sampling design when analyzing IAV datasets for reassortment.