A mallard mystery: Unraveling the genetic basis of green egg color

A series of experiments narrows the mystery down to one candidate gene.

You might not think about it when you prepare eggs for breakfast, but the color of an egg is an important ecological trait. The eggshell color is mainly determined by a mixture of three types of pigments: protoporphyrin-IX, biliverdin-IX and biliverdin zinc chelate. These pigments provide protection against damaging solar radiation and play a role in thermoregulation, creating the ideal conditions for embryonic development. Moreover, egg color is often a crucial factor in brood parasites that mimic the egg color of their unsuspecting hosts. In some species, the color of eggs is even used as a signal for female quality (see for example this blog post on hoopoes).

Despite this variety of roles in ecological and evolutionary processes, the genetic basis of egg color remains largely unknown. A recent study in the journal Molecular Ecology focused on the green egg color of a Chinese domesticated duck breed (the Jinding duck). What genes underlie this peculiar green color?

Comparing Genomes

The researchers performed a series of clever experiments to determine the genetic basis of green egg color (similar to the case of the mosaic canary described in this blog post). First, they compared the genomes of reciprocal crosses between Pekin ducks (with white eggs) and mallards (with green eggs). The genome-wide search identified a broad target region on chromosome 4 that was significantly associated with egg color. This region was explored in greater detail by analyzing the genomes of seven indigenous duck populations that differ in the coloration of their eggshells. The divergence between green-shelled and white-shelled populations could be traced to a small section of the target region, containing two genes: PRKG2 and ABCG2. The second gene (ABCG2) codes for a membrane transporter that carries biliverdin, one of the pigments that contribute to egg shell coloration. Sounds like the perfect candidate gene.

The genome-wide association study (GWAS) pointed to a region on chromosome 4 that might contain the genes underlying green egg color. From: Liu et al. (2021) Molecular Ecology.

Gene Expression

The researchers did not stop at identifying the candidate gene. They performed more detailed analyses to understand how this gene contributes to the green egg color. Eggshell formation takes place in the uterus, so the researchers measured gene expression in uterine tissues from four populations. As expected, ABCG2 was expressed at a higher level in the green-shelled groups compared to the white-shelled groups. Moreover, the data revealed that ABCG2 produces five distinct isoforms (i.e. different proteins that derive from the same DNA sequence). This is achieved by combining different sections of the gene during protein translation. In the mallard case, the third isoform (ABCG2-X3) is expressed most.

Finally, the researchers took a closer look at the DNA sequence of ABCG2. Does it contain genetic differences that clearly separate green-shelled from white-shelled ducks? Using a collection of sophisticated analyses (including ATAC-sequencing and a luciferase assay), the search could be narrowed down to one genetic variant on nucleotide 47,418,074 of chromosome 4. This position is targeted by different transcription factors (ATF and c/EBPα) in white-shelled compared to green-shelled ducks: ATF binds the white-shelled variant, whereas c/EBPα binds the green-shelled variant. The green egg color of the Jinding duck can thus be explained by a regulatory change in the expression of a specific isoform of the ABCG2-gene. The resulting protein is active in the uterus where it transports the pigment biliverdin from the blood onto the developing egg shell. Mystery solved.

A specific isoform of the candidate gene (ABCG2-X3) shows the highest expression level in green-shelled ducks. From: Liu et al. (2021) Molecular Ecology.

References

Liu et al. (2021). A single nucleotide polymorphism variant located in the cis‐regulatory region of the ABCG2 gene is associated with mallard egg colour. Molecular Ecology, 30(6), 1477-1491.

Featured image: Green eggs in the nest of a duck © Smudge9000 | Flickr

The role of hybridization in the domestication of geese

Gene flow patterns between wild and domestic geese changed during the domestication process.

Geese probably saved the Roman empire. The Gauls were secretly climbing the Capitoline Hill when they woke up a flock of geese. The noise of the honking geese alarmed the Romans that managed to fend off the Gaul attack. This story indicates that geese were already domesticated in Roman times. The earliest reliable reference to domestic geese can be traced back even further, to the 8th century BCE in Homer’s Odyssey. But when did humans domesticate geese?

This question is difficult to answer because different domestic goose breeds are derived from two species: the Greylag Goose (Anser anser) and the Swan Goose (Anser cygnoides). In addition, some breeds are probably the outcome of hybridization between these species, and several breeds are known to hybridize with their wild relatives. Marja Heikkinen and her colleagues tried to solve this complex puzzle of hybridization and domestication with genetic data. Their results recently appeared in the journal G3: Genes | Genomes | Genetics.

Gene Flow

The researchers collected samples from wild and domestic geese across Eurasia. Genomic data revealed a clear separation between wild Greylag Geese, European domestic breeds and Chinese domestic breeds. Demographic analyses suggested that the wild and domestic lineages diverged around 14,000 years BCE (although the authors indicate that this divergence time has wide confidence intervals and will need to be confirmed with more detailed analyses). This divergence was followed by several episodes of hybridization and consequent gene flow.

At the onset of domestication, gene flow was primarily from domestic into wild geese. Probably, geese were not intensively managed at that time, allowing domestic geese to interbreed with their wild relatives. Moreover, goose farmers might occasionally restock their flock by collecting eggs from the wild and raising them in captivity. By Medieval times, goose-keeping was a common phenomenon and the escaped birds would regularly mix with wild flocks. This resulted in gene flow in the opposite direction, from the wild into the domestic population. A patterns that is still visible in present-day goose populations.

A principal component analysis indicates a clear separation between wild Greylag Geese (blue), European domestic breeds (green) and Chinese domestic breeds (red). Some locations, such as Turkey, show admixture from different lineages.

Turkish Hybrids?

Interestingly, the amount of gene flow from wild into domestic goose populations differs between countries. In Finland and Norway – where goose rearing is not so popular – the genetic influence of wild birds is relatively low. In the Netherlands, however, the genetic signs of hybridization is more pronounced. This can be explained by the popularity of waterfowl collections in this country and the fact that many Greylag Geese winter in the Dutch fields (and a large proportion is even present year-round). There is thus ample opportunity for domestic geese to interbreed with wild ones.

Turkish goose populations showed genetic signatures from both European and Chinese domestic breeds. They might thus be a hybrid population between these independently domesticated breeds. Alternatively, the Turkish geese might represent the ancestral genetic variation of Greylag Geese, supplemented with some gene flow from Chinese breeds. More research is needed to solve this mystery and determine whether the domestication of geese started in Turkey.

References

Heikkinen, M. E., et al. (2020). Long-term reciprocal gene flow in wild and domestic geese reveals complex domestication history. G3: Genes, Genomes, Genetics, 10(9), 3061-3070.

Featured image: Domestic geese © Hippopx

This paper has been added to the Anseriformes page.

Why the “Red-breasted Meidum Goose” is probably not an extinct species

Convincing evidence to consider it an extinct species is currently lacking.

“Extraordinary claims require extraordinary evidence”, said Carl Sagan in the television program Cosmos. This statement came to mind when I read several news articles with bold titles, such as “4,600-Year-Old Egyptian Painting Depicts Extinct Species of Goose” and  “Ancient art reveals extinct goose.” These headlines refer to a recent study on the Meidum Geese, a 4,600-year-old Egyptian painting that historians described as “one of the great masterpieces of the Egyptian animal genre”. It depicts several goose species, including two Red-breased Geese (Branta ruficollis). However, a closer look at these colorful paintings reveals some striking differences with this well-known species. Could the Meidum Geese represent an extinct taxon?

 

Tobias Criteria

To answer this question, Anthony Romilio applied the Tobias criteria to the artwork. This method scores the characters between closely related taxa on a scale of dissimilarity. Low scores indicate that the taxa are very similar and could be classified as a single taxon (e.g., subspecies ), while high scores point to many dissimilarities and possibly distinct species. The first goose on the artwork showed low scores when compared to Greylag Goose (Anser anser) or Bean Goose (Anser fabalis), while the second painted goose most likely corresponds to a Greater White-fronted Goose (Anser albifrons). The third species of the Meidum Geese, however, did not accurately reflect Red-breasted Goose. Romilio writes that “This raises the possibility that the ‘Medium Geese 3′ may illustrate a distinct, yet now extinct goose population as has been suggested previously.”

The Meidum Geese. The top left and bottom right paintings could correspond to Greylag Goose of Bean Goose. The pair in the top right are most likely Greater White-fronted Geese. But what about the Red-breased Geese?

 

Counterarguments

Stating that there was an extinct goose species in ancient Egypt based on a painting is quite an extraordinary claim. In contrast to the news headlines, the paper provides a very balanced analysis of this claim. And although it is a possibility, I would argue that the extraordinary evidence to back this claim is currently missing. I am not convinced for two main reasons:

1. Unique Artwork. The Meidum Geese are the only depiction of a Red-breased Goose in Egyptian art (to my knowledge). This suggests that they might have been seen as rare vagrants, and were later painted from the artists’ memory which can explain the differences. Moreover, if the painting represented an extinct taxon, you could expect independent artwork depicting this colorful bird.
2. Not Accurate Enough. In the paper, the author argues that other species are realistically painted and hence we can assume that the “Red-breasted Meidum Goose” is also realistic. First, this reasoning is contradicted by the analysis of Meidum Goose 1, which could be either a Greylag Goose or a Bean Goose. If the paintings were accurate, you should be able to tell the difference. And second, the other goose species on the artwork belong to the genus Anser, which is not known for its colorful members. These “grey geese” do not leave much room for artistic freedom, in contrast to the colorful patterns on the Red-breasted Goose.

 

Fossils and Genomes

What could convince me that the “Red-breasted Meidum Goose” does represent an extinct species? Another clue might come from the fossil record or genetic analyses. Recently, a goose skull was discovered on Crete that is similar to the Red-breasted Goose. A morphological analysis of this skull might reveal whether it belonged to an unknown species that is closely related to the Red-breasted Goose. In addition, researchers might be able to extract ancient DNA from the skull and perform a proper phylogenetic analysis.

The genomes of present-day goose species might also hold the key to this mystery. Genomic analyses have uncovered signatures of hybridization events with now extinct species in the genomes of extant species, a phenomenon known as ghost introgression. Given the high levels of hybridization in geese, the extinct Egyptian species might have interbred with the Red-breasted Goose or another goose species. If so, we might be able find genetic traces of these ancient hybridization events. And if we are lucky, the introgressed regions might provide some insights into the phenotype of this extinct species. I am aware that this is very speculative and wishful thinking. But it would certainly be extraordinary evidence!

 

A Hybrid Goose?

In a discussion on Twitter, someone proposed the possibility of a hybrid. Although I like this suggestion, I do not think it is likely. If it was a hybrid, there was clearly a Red-breasted Goose involved. And hybrids with this species tend to be “drab” and lose their bright red markings.

A putative hybrid between Red-breasted Goose and Barnacle Goose. © Dave Appleton | Flickr.

 

References

Romilio, A. (2021). Assessing ‘Meidum Geese’ species identification with the ‘Tobias criteria’, Journal of Archaeological Science: Reports 36:102834.