Pigment-based coloration is a common trait found in a variety of organisms across the tree of life. For example, calcareous avian eggs are natural structures that vary greatly in color, yet just a handful of tetrapyrrole pigment compounds are responsible for generating this myriad of colors. To fully understand the diversity and constraints shaping nature's palette, it is imperative to characterize the similarities and differences in the types of compounds involved in color production across diverse lineages. Pigment composition was investigated in eggshells of eleven paleognath bird taxa, covering several extinct and extant lineages, and shells of four extant species of mollusks. Birds and mollusks are two distantly related, calcareous shell-building groups, thus characterization of pigments in their calcareous structures would provide insights to whether similar compounds are found in different phyla (Chordata and Mollusca). An ethylenediaminetetraacetic acid (EDTA) extraction protocol was used to analyze the presence and concentration of biliverdin and protoporphyrin, two known and ubiquitous tetrapyrrole avian eggshell pigments, in all avian and molluscan samples. Biliverdin was solely detected in birds, including the colorful eggshells of four tinamou species. In contrast, protoporphyrin was detected in both the eggshells of several avian species and in the shells of all mollusks. These findings support previous hypotheses about the ubiquitous deposition of tetrapyrroles in the eggshells of various bird lineages and provide evidence for its presence also across distantly related animal taxa.

Department of Chemistry Hunter College Belfer Research Building City University of New York New York New York United States of America

Department of Psychology University of Massachusetts Amherst Massachusetts United States of America

Department of Zoology and Laboratory of Ornithology Palacký University Olomouc Czech Republic

The Graduate Center City University of New York New York New York United States of America Department of Psychology Hunter College City University of New York New York New York United States of America

The Graduate Center City University of New York New York New York United States of America Division of Invertebrate Zoology American Museum of Natural History New York New York United States of America

The Graduate Center City University of New York New York New York United States of America Division of Invertebrate Zoology American Museum of Natural History New York New York United States of America Department of Chemistry Hunter College Belfer Research Building City University of New York New York New York United States of America

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