Scientists’ Colorful Quest To Discover How Parrots Became Green | @GrrlScientist

A team of scientists developed a methodology to map out molecular processes and used that to identify the mysterious yellow pigment gene that parrots use to create their iconic green plumage

by GrrlScientist for Forbes | @GrrlScientist

This piece was a Forbes’ Editor’s Pick.

An adult male “wild type” (green) budgerigar (Melopsittacus undulatus).
(Credit: public domain.)

Many birds’ plumage colors are somehow derived from the foods they eat (for example, read this) — but parrots aren’t like most birds. A recently published study has identified the gene encoding the enzyme that creates yellow pigment in budgerigars. Yellow pigment is half of the yellow + blue equation necessary to make green plumage color in parrots. When the yellow gene is deactivated, parrots’ plumage becomes blue. The clever stepwise methodology that the researchers developed to specifically identify the budgerigar’s “yellow gene” was originally devised with an eye towards adapting it to identify genetic and biochemical processes that impact human health.

Budgerigars have a plethora of “color-coded” genes

The normal wild — “wild type” — budgerigar, Melopsittacus undulatus, is a small green parrot with a yellow face, a long pointed tail, and charcoal grey feathers with delicate yellow edging on its back and wings. These popular parrots are also known as the shell parakeet or simply as the “budgie” or “parakeet”. Hardy, with outsized personalities, and easy to breed in captivity, budgerigars have been kept as pets and as avicultural breeding subjects for longer than one century. As a result, a variety of plumage colors and patterns have been developed in captive birds over the decades. In fact, most people who go into a pet shop may not know what a wild budgerigar looks like because they are confronted with flocks of domesticated budgies that come in a veritable rainbow of colors and color patterns. Extensive studies of budgerigar inheritance patterns have provided bird breeders with a profound working knowledge of the genetics of plumage color for budgies and other green parrot species long before scientists figured out what a gene actually is.

“Budgerigars are a great system for studying parrot colors because artificial selection over the last 150 years has resulted in a large number of simple Mendelian genetic traits that affect color,” said the study’s first author, Thomas Cooke, who was a graduate student in genetics at Stanford University when he did this work. (Dr. Cooke is now at the Massachusetts Institute of Technology, where he is beginning a postdoctoral fellowship.)

This yellow pigment is a “psittacofulvin”, and it’s special because it’s only found in parrots. Psittacofulvins comprise a group of structurally related pigments that give parrots their stunning variety of brilliant red, orange, and yellow colors. Not only are these pigments pretty, but they’re functional: a few years ago, a study found that feathers colored with psittacofulvin pigments resist degradation by bacteria better than white ones (ref; read more.)

Basically, these genes are “color-coded”. In budgerigars, the blue trait is co-expressed alongside the yellow trait so together, they combine to create green plumage. But when expression of the yellow pigment is lost, all green feathers in a “wild type” budgie are replaced with blue and yellow feathers become white.

Blue and green budgerigars (Melopsittacus undulatus). A budgerigar with blue plumage cannot produce yellow pigment. (Credit: Holly Clark / CC BY-NC-ND 2.0)

Although the genetics of plumage color is well established in budgerigars, the molecular and biochemical aspects underlying plumage color remain largely unexplored. For example, more than one hundred years of genetic studies confirm that the budgerigar’s yellow psittacofulvin pigment gene is a simple Mendelian trait that is controlled by just one locus — one gene — in the genome, that follows a simple “yes-you-have-it/no-you-don’t-have-it” inheritance pattern that is clearly visible. For this reason, Dr. Cooke proposed that it should be straightforward to track down precisely which gene it is.

But how can one specific gene that governs just one visible trait be distinguished from the millions of other genes contained within an organism’s entire genome? On the surface, this seems more daunting than searching for the proverbial needle in a haystack. But Dr. Cooke and his colleagues were equal to the task. Dr. Cooke’s research bridges the gap between molecular biology and biochemistry to investigate pigmentation in “non-traditional research animals” and to use that as a pathway towards unlocking the secrets of cellular and developmental processes. His goal was to develop a master plan to diagnose how biochemical mechanisms lead to variations in the physical appearance, or phenotype, of an individual. With these goals in mind, Dr. Cooke assembled a team of researchers who helped him develop a viable step-by-step strategy for hunting down and identifying the budgerigar’s elusive yellow psittacofulvin gene.

Overlapping regions of the genome pointed towards the yellow gene

Budgerigars are a wonderful system to develop methodologies for genetic detective work. For example, all blue budgerigars are descendants of a single bird that was born around 130 years ago. Thus, all blue budgerigars share the same mutated psittacofulvin gene. This allowed Dr. Cooke and his colleagues to search through the entire budgerigar genome for regions that were (1) shared only by blue budgies, and (2) differed between wild type (green), and derived (blue), budgies. Their search uncovered eleven candidate genes located next to each other in one region (Figure 3):

Figure 3. Haplotypes Associated with the blue Phenotype
(A) Haplotypes for 63 SNPs in a 3 Mb region centered on the blue locus association peak were inferred with the software program PHASE. At a subset of these loci, outlined in gray, all blue individuals carried the same haplotype (the two flanking SNPs are also included in this set). Population-wide haplotype counts were calculated from the most likely pairs of haplotypes carried by each individual and their associated probabilities. Only haplotypes with greater than or equal to 2 counts are shown, except haplotype 8, which was found in only one sample but shows evidence of an ancestral recombination between SNPs at 21,019,187 and 21,161,723. The ancestral alleles were determined by whole-genome sequence alignment to 14 other avian species.
(B) RefSeq gene models and descriptions for genes located within the blue-shared haplotype, with positions of SNPs shown, including the two flanking SNPs. (doi:
10.1016/j.cell.2017.08.016)

Dr. Cooke and his colleagues searched the online gene database and tentatively identified these 11 candidate genes from short sequences contained within each. One candidate gene looked particularly promising because its sequence was similar to another gene, now known in the literature as MuPKS, that encodes an enzyme that synthesizes yellow pigments in bacteria and fungi. (MuPKS is a shortened version of its formal name, Melopsittacus undulatus polyketide synthase.) Could this be the yellow psittacofulvin gene that Dr. Cooke and his colleagues were looking for?

The yellow gene is highly expressed in growing feathers

An intriguing clue about where in the budgie’s body that this gene is most highly expressed was provided by a strange pet shop budgerigar known as “Twinzy”. This bird resulted from a rare developmental accident where two different colored twins fused into one individual. In the case of Twinzy, this accident occurred very early in development, so he looks like two different budgerigars had been neatly sewn together to create one individual — which is a good way to think about Twinzy (read more).

Bilateral gynandromorph budgerigar named “Twinzy”. This bird is the result of developmental fusion between derived (blue) and wild type (green) budgerigar twins. The blue budgerigar results from the loss of the yellow psittacofulvin pigment.
(Youtube screengrab.)

The near-perfect symmetry of Twinzy’s blue and green halves suggested to the team that high levels of the yellow psittacofulvin pigment is synthesized locally — probably within the feather itself — and that the pigment is most highly expressed during molt; when new feathers are actively growing. Thus, searching for the complete MuPKS transcript would be easiest and most efficient if the researchers looked for it in actively growing feathers.

The MuPKS “yellow gene” encodes an enzyme found from birds to bacteria

Why do blue budgerigars lose their yellow psittacofulvin pigment? Is this due to blocked production of the gene, or is it due to a mutation that prevents the resulting protein from working properly?

Dr. Cooke and his colleagues answered this question by looking for MuPKS gene transcripts in regenerating feathers from both wild type green and derived blue budgerigars. They found that both green and blue budgerigars showed similar levels of MuPKS expression. This indicated that nothing was preventing the yellow psittacofulvin pigment gene from being produced so there had to be some sort of change to the gene product (an enzyme) that prevented it from doing its job properly.

Dr. Cooke and his colleagues scrutinized the MuPKS protein’s sequence of amino acids from blue budgies to find any tiny changes that were not present in wild type green budgerigars — and they discovered just one change. All blue birds had two copies of this mutated MuPKS protein, both of which had the same alteration in the same place in its sequence (Figure 5):

Figure 5. Variant in the gene sequence that encodes the budgerigar yellow psittacofulvin pigment that deactivates its biosynthetic activity. (doi:10.1016/j.cell.2017.08.016)

Did all their study birds show this same change? Dr. Cooke and his colleagues examined both copies of MuPKS in all 162 of their wild type green study birds and compared them to both copies of MuPKS in their 118 derived blue study birds. They also sequenced 15 museum specimens from Australia. They found the blue birds all had the altered MuPKS (Figure 5A). Further, when they aligned the budgerigar MuPKS protein’s amino acid sequences with the synonymous protein’s amino acid sequences from other organisms, they found that one particular animo acid (dubbed “R644”) is identical in functional versions of this protein whether it is found in vertebrates, fungi or even in bacteria (Figure 5B). This animo acid is strategically located in the “active site” of the enzyme, which is critically important for manufacturing the yellow psittacofulvin pigment (Figure 5D). When this one amino acid residue is altered, the enzyme cannot work properly, and the yellow psittacofulvin pigment is not synthesized.

MuPKS is an ancient gene that parrots re-purposed for a new evolutionary function

Now that Dr. Cooke and his colleagues had successfully mapped the location of the candidate gene in the budgerigar genome, analyzed its sequence and identified how the resulting enzyme could be deactivated, they were fairly certain that this was the source of the yellow psittacofulvin pigment in parrots. But they still had to prove it. So Dr. Cooke and his colleagues copied the budgerigar MuPKS gene, placed it into yeast, and cultured these wee beasties to see if they would produce the budgerigar’s yellow psittacofulvin pigment. They did.

Dr. Cooke and his colleagues then looked more closely at the many other organisms that have MuPKS in their genomes, including snakes, lizards and ray-finned fishes, to track this gene’s evolutionary history and origins. The team aligned copies of MuPKS gene sequences and used those to construct a phylogenetic family tree (Figure 7):

Figure 7. Phylogeny of Metazoan Polyketide Synthases
(A) Maximum likelihood tree based on an alignment of the KS domains of metazoan polyketide synthases, fatty-acid synthases, and their homologs in fungi, eukaryotic outgroups, and bacteria. Bootstrap values (based on 1,000 replicates) are indicated at the tree nodes. The scale bar below the tree denotes sub- stitutions per site. Species are colored according to the clades shown in ©. The tree is rooted by the outgroup mycocerosic acid synthase (MAS) from Mycobacterium.
(B) Domain structures for the enzymes shown in (A). Colors denote domains commonly found in polyketide synthases, fatty-acid synthases, and non-ribosomal peptide synthases. Inactive pseudo-domains, or domains likely to be inactive based on sequence features, are denoted by ‘‘J’’ (KS, MAT, and ACP make up the minimal set of domains for a functional polyketide synthase). Partial sequences or those containing probable artifacts from genome assembly errors are left blank. © Cladogram for species shown in (A).
(doi:10.1016/j.cell.2017.08.016)

This MuPKS family tree shows the vertebrate versions of this gene all look very similar and they even have similar neighboring genes, indicating that they came from the same ancestral gene. Thus, the research team concluded that MuPKS’s regulation pattern had been altered in green parrots so it ended up being expressed in growing feathers, thereby creating yellow plumage pigment.

“Presumably the gene has some function in non-parrots besides pigmentation, but we don’t know what that might be,” Dr. Cooke said. He noted that similar MuPKS genes are found in almost all birds, but birds outside the parrot family, such as chickens and crows, don’t express the enzyme in their feathers, and so they aren’t yellow.

“Non traditional research organisms”, like parrots, can teach scientists a lot about biology

“What Thomas conceptually demonstrated was we could go into any organism” and learn something interesting and useful about its biochemistry, according to the study’s senior author, Carlos Bustamante, professor of biomedical data science and of genetics at Stanford.

“We identified an uncharacterized gene in budgerigars that is highly expressed in growing feathers and is capable of synthesizing the budgie’s yellow pigments,” Dr. Cooke said.

Most exciting to me is that these studies will shed light on parrots’ special psittacofulvin pigments, and how the enzymes that produce those pigments evolved. This work will also add an important dimension to evolutionary and ecological studies of parrots because these birds rely upon plumage color to help them choose mates.

“It would be interesting to see what sorts of changes at the DNA level underlie coloration differences within and between different species of parrots,” Dr. Cooke added.

The methodical and multidisciplinary approach that Dr. Cooke and his colleagues developed for hunting down and characterizing the molecular biology and biochemistry of the yellow psittacofulvin pigment can be adapted by medicine, where it will provide medical researchers with an important roadmap for hunting down and characterizing the next key medicinal compound or biochemical pathway that affects and can improve human health.

“It really demonstrates the power of emerging model systems,” Professor Bustamante said.

Source:

Thomas F. Cooke, Curt R. Fischer, Ping Wu, Ting-Xin Jiang, Kathleen T. Xie, James Kuo, Elizabeth Doctorov, Ashley Zehnder, Chaitan Khosla, Cheng-Ming Chuong, and Carlos D. Bustamante (2017). Genetic Mapping and Biochemical Basis of Yellow Feather Pigmentation in Budgerigars, Cell, published online on 5 October 2017 before print | doi:10.1016/j.cell.2017.08.016

Also cited:

Edward H. Burtt, Jr, Max R. Schroeder, Lauren A. Smith, Jenna E. Sroka, and Kevin J. McGraw (2011). Colourful parrot feathers resist bacterial degradation, Biology Letters, 7:214–216 | doi:10.1098/rsbl.2010.0716


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Originally published at Forbes on 13 November 2017.

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