Ortho2Web: a workflow for disentangling the roles of hybridization and allopolyploidization in reticulation within Campanulaceae

Abstract

Background: Phylogenetic networks (Web of Life) depict the intricate evolutionary dynamics of organisms more accurately than simple bifurcating trees (Tree of Life), because processes such as hybridization and polyploidy connect lineages through reticulation. However, most existing workflows still examine evolutionary mechanisms separately. To provide an integrated view, we studied the bellflower tribe (Campanuleae), a lineage with a well‑documented reticulate past, and developed Ortho2Web, a workflow that teases apart hybridization and polyploidization within a single analytical framework. Results: Ortho2Web employs multiple strategies to reduce the impact of non-biological factors, including (1) integrating multi-source genomic data to minimize sampling gaps, (2) generating complementary datasets to quantify phylogenetic signals, and (3) using tree-based orthology inference methods to mitigate the effects of paralogs. It then focused on clarifying the roles of biological factors, such as incomplete lineage sorting (ILS), hybridization/gene flow, and polyploidization. When applied to Campanuleae, the workflow produced a robust phylogenetic backbone and showed that early diversification was driven by interacting hybridization and allopolyploidization, while ILS contributed only marginally. Conclusions: By integrating diverse data sources and analytical approaches in a modular, scalable framework, Ortho2Web offers a practical platform for inferring web-like phylogenies and elucidating the mechanisms of reticulate evolution. The Ortho2Web workflow is freely available at: https://github.com/PhyloAI/Ortho2Web.