The blue-green eggs of dinosaurs: How fossil metabolites provide insights into the evolution of bird reproduction

Open-nesting birds use biological pigments in eggshell to camouflage their unhatched offspring, varying the colour to account for the nesting environment and location. The tetrapyrrolic pigments protoporphyrin (PP) and biliverdin (BV), which both participate in the haem metabolism, are responsible for the reddish brown of chicken eggs and the brilliant blue of robin and emu eggs. However, eggshell pigmentation correlates with the nest type in a wide range of avian species and suggests that coloured eggs are basal to the avian lineage, extending back to their non-avian dinosaur origins. Detecting preserved eggshell pigments could thus shed light on dinosaur nesting behaviour. Using HPLC separation coupled to ESI-Q-TOF mass spectrometry, we here provide the first record of the eggshell pigments PP and BV preserved in fossils from three different localities, in 66 million year-old oviraptorid eggshell ( Macroolithus yaotunensis ). These eggs were presumably laid in at least partially open nests by the oviraptorid Heyuannia huangi and camouflaged by an originally blue-greenish egg colouration. Such a blue-greenish eggshell pigmentation hints at increased paternal care in Heyuannia . Shell porosity measurements, preserved clutches and parental animals support an open nesting behaviour for oviraptorid dinosaurs. Furthermore, the detection of PP, together with supporting microscopic observations, represents the first evidence for cuticle preservation in fossil eggshell. Our study demonstrates that molecular biomarkers, such as preserved metabolites, can be used to trace the evolution of modern avian traits, and to provide insights into dinosaur reproductive biology and the preservation of endogenous organic matter in fossil vertebrates.