Persistent conflict in palaeognath phylogeny revealed by quartet-based and ML analyses

The evolutionary relationships among palaeognath birds remain contentious despite extensive phylogenomic analyses. While the ostrich is consistently identified as the earliest diverging lineage, the relationships among non-ostrich palaeognaths remain unresolved, with conflicting topologies and low support across different genomic datasets. In this study, we reanalyzed three genomic marker sets (CNEEs, UCEs, and INTRONs) using alternative maximum-likelihood (ML) approaches and the quartet-based SeaLion method, which assesses phylogenetic signal by evaluating split-pattern information in polarized quartets.

Our results strongly support ostrich as the first split and reinforce ratite paraphyly. However, non-ostrich relationships remain ambiguous, with different best-supported trees emerging across datasets. Among them, the UCEs dataset provided the strongest signal, consistently supporting a "tinamou-first" scenario with the clade tinamou + moa emerging as sister clade to all other non-ostrich palaeognaths while rhea and kiwi form the most distinct sister-pair. Despite applying quartet-based filtering to reduce phylogenetic noise, substantial conflict persisted, indicating that weak internal branch signal rather than methodological biases alone underlies the lack of resolution.

Our results suggest that the unresolved relationships among non-ostrich palaeognaths are driven by intrinsic data limitations rather than analytical shortcomings. Very short internal branches and extensive incomplete lineage sorting indicate that these divergences may reflect a true polytomy. While this scenario is proposed for Neoaves, a similar process may underlie the persistent uncertainty in palaeognaths. Future progress will depend on expanding comparative frameworks with additional high-quality outgroup genomes, providing a stronger basis for quartet-based phylogenetic inference.