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Abstract

Coloration and patterning have been implicated in lineage diversification across various taxa, as color traits are heavily influenced by sexual and natural selection. Investigating the biochemical and genomic foundations of these traits therefore provides deeper insights into the interplay between genetics, ecology, and social interactions in shaping the diversity of life. In this study, we assessed the pigment chemistries and genomic underpinnings of carotenoid color variation in naturally hybridizing Dinopium flamebacks in tropical South Asia. We employed reflectance spectrometric analysis to quantify species-specific plumage coloration, High-Performance Liquid Chromatography (HPLC) to elucidate the feather carotenoids of flamebacks across the hybrid zone, and Genome-Wide Association Study (GWAS) using next-generation sequencing data to uncover the genetic factors underlying carotenoid color variation in flamebacks. Our analysis revealed that the red mantle feathers of D. psarodes primarily contain astaxanthin, with small amounts of other 4-keto-carotenoids. In contrast, the yellow mantle feathers of D. benghalense predominantly contained lutein and 3'-dehydro-lutein, alongside minor amounts of zeaxanthin, β-cryptoxanthin, and canary-xanthophylls A and B. Hybrids with an intermediate, orange, coloration deposited all of these pigments in their mantle feathers, with notably higher concentrations of carotenoids with ε-end rings. The GWAS analysis identified the CYP2J2 gene, which plays a role in carotenoid ketolation, as associated with the expression of carotenoid coloration. This gene exhibited significant allele variation and evidence of multiple copies in flamebacks. These findings contribute to the growing knowledge of avian carotenoid metabolism and highlight how genomic architecture can influence phenotypic diversity.