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. 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 co-varying with color. This gene exhibited significant allele variation and evidence of multiple copies across species. These findings contribute to the growing knowledge on avian carotenoid metabolism and highlight how genomic architecture can influence phenotypic diversity.

Island ecosystems have emerged as vital model systems for evolutionary and speciation studies due to their unique environmental conditions and biodiversity. This study investigates the population divergence, hybridization dynamics, and evolutionary history of hybridizing golden-backed and red-backed Dinopium flameback woodpeckers on the island of Sri Lanka, providing insights into speciation processes within an island biogeographic context. Utilizing genomic analysis based on next-generation sequencing, we revealed that the Dinopium hybrid zone on this island is a complex three-way hybrid zone involving three genetically distinct populations: two cryptic populations of golden-backed D. benghalense in the north and one island-endemic red-backed population of D. psarodes in the south of Sri Lanka. Our findings indicate asymmetric introgressive hybridization, where alleles from the southern D. psarodes introgress into the northern D. benghalense genome while phenotype remains adapted to their respective northern arid and southern wet habitats. The discovery of two genetically distinct but phenotypically similar D. benghalense populations in northern Sri Lanka highlights the process of cryptic speciation within island ecosystems. These populations trace their ancestry back to a common ancestor, similar to the Indian form D. b. tehminae, which colonized Sri Lanka from mainland India during the late Pleistocene. Subsequent divergence within the island, driven by selection, isolation-by-distance, and genetic drift, led to the current three populations. Our findings provide evidence of cryptic speciation and within-island population divergence, highlighting the complexity of hybridization and speciation processes. These findings further emphasize the intricate nature of evolutionary dynamics in island ecosystems.