Supplementary data: Phylogenomic and comparative genomic analyses support a single evolutionary origin of flatfish asymmetry

The eye migration that characterizes flatfish cranial development provides a unique opportunity to study the molecular mechanisms underlying anatomical asymmetry. In a paper in Nature Genetics, Lü et al. reported a comparative genomic assessment of flatfish asymmetry and claimed that the two major lineages (Pleuronectoidei and Psettodoidei) are polyphyletic, each evolving asymmetric bodies convergently from different symmetric ancestors (flatfish polyphyly). Here we revisit this finding by analyzing three independent genome-scale datasets, showing that support for flatfish polyphyly results from a failure to accommodate lineage-specific variation in base composition. We also re-analyzed a genomic dataset to identify positively selected genes (PSGs) but using instead an inferred tree that groups flatfishes as monophyletic, implying a single evolutionary origin of the asymmetric body plan. These results suggest a key evolutionary role of thyroid hormones (THs) and bone morphogenetic proteins (BMPs), bridging evidence from the fossil record and single-species molecular assays.