HPLC and Genomics Uncover How Woodpeckers Get Their Color

A joint study between the University of British Columbia (Vancouver, British Columbia, Canada), the Royal Alberta Museum (Edmonton, Alberta, Canada).and the University of Colombo (Colombo, Sri Lanka) employed reflectance spectrometric analysis to quantify species-specific plumage coloration, and high performance liquid chromatography (HPLC) to elucidate the feather carotenoids of South Asian flameback woodpeckers to assess their pigment chemistries and genomic underpinnings. A paper based on this research was published in Molecular Ecology (1).

The naturally hybridizing Dinopium flameback woodpeckers of Sri Lanka provide an excellent source of biochemical and genetic materials for the investigation of color expression. Sri Lanka is the habitat of two species of Dinopium flamebacks: the endemic D. psarodes (red-backed flameback), known for its striking crimson-red coloration on the mantle, coverts, secondaries and head; and D. benghalense (Black-rumped flameback), which displays golden-yellow coloration on the mantle, coverts, and secondaries, along with a crimson-red head (2–4).

These two species hybridize frequently in locations where they share a habitat, and this hybridization has resulted in woodpeckers that exhibit a range of intermediate orange hues on the mantle, coverts and secondaries, while maintaining the crimson-red head (4,5). However, the two species show little genetic differentiation between them (6). This natural gradient of plumage coloration from red to orange to yellow, combined with extensive genomic admixture and shallow genetic differentiation offers an ideal system to examine the genetic basis of carotenoid color expression (6). In addition, the recent discovery of cryptic divergence within the yellow-backed D. benghalense insinuates three-way hybridization among Dinopium flamebacks on the island (6). This discovery further enhances system's potential for examining the genetic architecture of red versus yellow carotenoid coloration (1).

In this study, the team captured red-backed D. psarodes, yellow-backed D. benghalense and orange-backed intermediate flameback woodpeckers across the Dinopium hybrid zone in Sri Lanka (3,4,6) using mist nets, with the aid of lifelike wooden-crafted flameback decoys and playbacks of locally recorded flameback vocalizations to attract individuals into the nets. From each captured bird, the team of scientists collected approximately 50 μL of blood as well as three crown feathers and three mantle feathers, which were placed in brown envelopes and stored in the refrigerator until pigment extraction. In total, mantle feathers from 40 flamebacks (13 red-backed D. psarodes, 19 yellow-backed D. benghalense, and eight orange-backed intermediate-colored flamebacks) and red crown feathers from 20 birds were obtained (1).

The team’s analysis showed that the red mantle feathers of D. psarodes primarily contained astaxanthin, with small amounts of other 4-keto-carotenoids, while 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 (1).

The findings indicate that the carotenoid color expression of the flameback woodpecker is the result of a complex interplay between carotenoid metabolism, stress response pathways and potential regulatory effects on carotenoid expression. The observed differences in mantle coloration, primarily reflected in hue, may highlight species-specific adaptations to distinct ecological environments, while the conserved red crown coloration across woodpecker lineages likely serve a consistent functional or signaling role. The identification of specific genes through a genome-wide association study (GWAS) admixture analysis emphasizes the multifaceted role of genetic factors in carotenoid bioconversion and color expression in these birds. Collectively, the team believes that these insights heighten our understanding of the genetic and biochemical mechanisms driving carotenoid color variation in this unique woodpecker species and opens the door for continued research into the evolutionary and ecological significance of these traits (1).

References

1. Ranasinghe, R. W.; Hudon, J.; Seneviratne, S. S, et al. Biochemical and Genomic Underpinnings of Carotenoid Colour Variation Across a Hybrid Zone Between South Asian Flameback Woodpeckers. Mol. Ecol. 2025, e70084. DOI: 10.1111/mec.70084
2. Del Hoyo, J.; Del Hoyo, J.; Elliott, A. et al. Editors; Handbook of the Birds of the World, Vol. 7; Lynx Edicions, 2002.
3. Fernando, S. P.; Irwin, D. E.; Seneviratne, S. S. Phenotypic and Genetic Analysis Support Distinct Species Status of the Red-Backed Woodpecker (Lesser Sri Lanka Flameback: Dinopium psarodes) of Sri Lanka, The Auk 2016, 133 (1), 497–511. DOI: 10.1642/AUK-15-233.1
4. Fernando, S.; Seneviratne, S. Quantitative Analysis of the Variation of Plumage Colouration in Dinopium Flame-Back Complex of Sri Lanka. Wild 2015, 3, 1-7.
5. Freed, L. A.; Warakagoda, D.; Cann, R. L. et al. A Hybrid Swarm of Dinopium Woodpeckers in Sri Lanka. The Wilson Journal of Ornithology 2015, 127 (1), 13-20. DOI: 10.1676/14-065.1
6. Ranasinghe, R. W.; Seneviratne, S. S.; Irwin, D. Cryptic Hybridization Dynamics in a Three-Way Hybrid Zone of Dinopium Flamebacks on a Tropical Island. Ecol. Evol. 2024, 14 (12), e70716. DOI: 10.1002/ece3.70716