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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.