News Release 

Genomic insights: How female butterflies alter investment in attractiveness vs. fecundity

By analyzing the genomes of orange and white females of the clouded yellow butterfly, researchers have identified the location and key components of an alternative life history switch that is only expressed in females

Stockholm University

Have you ever wandered through your garden or a meadow on a summer day and wondered why and how butterflies exhibit such beautiful and diverse colors? Scientists have, especially when thinking about butterflies in the genus Colias. In most Colias butterflies, all males and most females are an orange or yellow color, but some females are white. Rare white forms are common in many animals, however, in some Colias butterflies, these white forms, called Alba, are commonly found in every generation, making up 5 % to sometimes 30% of females. This is unexpected because orange/yellow wing color is an important signal for mate recognition, thus Alba females should be at a disadvantage. For nearly a century, scientists have been working to understand why and how Alba females arise and are maintained within butterfly populations.

Studies over the past century have revealed that Alba is not just a change in wing color, but also a visual manifestation of an alternative life history strategy. A life history strategy is a pattern of co-evolved life history traits (for example number of offspring, size of offspring, and lifespan) and these strategies are shaped by the way individuals allocate limited resources to the different traits that affect their Darwinian fitness. Previous work in Colias found that orange females invest their resources in synthesizing colored pigments, while Alba females reallocate these resources away from the production of colored pigments and into other developmental processes. Presumably as a result of this reallocation, Alba females benefit from a higher fat content and fecundity compared to orange females. However, males prefer to mate with orange females, which should be a cost to Alba. Although studies throughout the 80's and 90's worked to understand more about the biotic and abiotic factors that maintained the color polymorphism in the wild, the genetic basis of Alba has remained unknown. Seeking to understand the mechanisms that give rise to Alba, a team of evolutionary biologists has focused their attention upon the mechanism of Alba using a diverse set of research approaches.

"Life history tradeoffs are found across the tree of life, yet we know very little about their genetic basis and how they evolve. This is especially true for female-limited tradeoffs; essentially nothing is known", said senior author Christopher Wheat, Associate Professor of Population Genetics at Stockholm University.

In order to identify the genomic region causing Alba, the researchers first had to make a genome for the clouded yellow butterfly (Colias crocea), which is common throughout Europe and neighboring regions. They then sequenced many individual genomes of wild caught orange and Alba females, as well as offspring from crosses between family lines carrying the two different color morphs. Analyzing this data, they were able to identify the single genomic region associated with the color difference. In that region, they found a transposable element insertion that was unique to Alba females. This insertion was located near a gene called BarH-1, which encodes a homeobox transcription factor. Homeobox transcription factors play important roles during embryonic development.

"BarH-1 was a really exciting candidate gene for Alba because in the fruit fly Drosophila melanogaster BarH-1 plays a role in pigment granule development in their eyes. I had previously found that Alba butterflies have significantly less pigment granules in their wing scales compared to orange females. In Pierid butterflies these granules contain the pigments and when granules are removed, Colias wings turn white", said lead author Dr. Alyssa Woronik, whose PhD at Stockholm University was focused upon studying and finding the basis of Alba.

However, the researchers wanted to do more than just show a significant association between Alba and insertion region of the genome near BarH-1. Therefore, they used antibody staining to detect whether BarH-1 was present in developing wings, as it has never been reported there before. This approach revealed that early during pupal development, the BarH-1 protein is present in the cells that build wing scales in Alba females, but not in orange. These results suggested that BarH-1 might play a role in the orange to white wing color change by suppressing the formation of pigment granule. To test this functional prediction, they used CRISPR/Cas9 gene editing to generate mosaic BarH-1 knockouts. A mosaic knockout means that some cells within an individual had a functional copy of the BarH-1 gene, while others did not. In Drosophila, normal BarH-1 function results in red eye pigments, while BarH-1 knockouts have white eyes where there is no pigment. Unlike flies, normal Colias eyes are emerald green. Interestingly, mosaic BarH-1 knockout individuals of Colias have a mosaic of green and black in their eyes, with green being cells with functional BarH-1, while black color arises due to BarH-1 knockout. Green and black eye color mosaics told the researchers their BarH-1 knockouts were working. Furthermore, Alba females with mosaic eyes also had white and orange mosaic wings. The orange regions, atypical of normal Alba females, were presumed to arise from cells lacking a functional copy of BarH-1. Further examination of these mosaic individuals revealed that the orange scales had significantly more pigment granules than white scales, similar to the wing scales of normal orange animals. However, neither males nor orange females with mosaic eyes had any mosaic color effects on their wings. These findings supported the prediction that BarH-1 expression in wing scale building cells gives rise to the white color by suppressing the formation of pigment granules, a function that does not happen in males or orange females.

There are about 90 species of Colias butterflies worldwide and they can be found on every continent except Australia and Antarctica. About 30% of the species exhibit polymorphic females and previous research suggested that the same mechanism gives rise to Alba across all species. To test this prediction, the researchers checked whether Alba females in the North American species Colias eurytheme also had less pigment granules than orange females. They did, suggesting that suppression of pigment granule formation is not limited to C. crocea. Additional work showed that Alba females had more abdominal fat stores than orange, consistent with previous studies in C. eurytheme. These results suggest the intriguing possibility that the cause of Alba across all Colias species might be the same, but this requires further study in more species.

Based on these findings, the working hypothesis of the research team is that the Alba tradeoff arises via a simple Y-model of reallocation where reduced pigment granule formation results in reduced pigment synthesis. This in turn leaves more resources free to be used for other developmental processes within the energetically closed system of the developing pupa. However, it is possible that the Alba-associated physiological components are also affected by expression of BarH-1, or other genes surrounding the insertion, in other tissues or at other times in development. Alternatively, other mutations, located near the insertion, may cause these traits. Testing these alternative hypotheses, as well as determining whether Alba has a single genetic basis among species in the Colias genus, is the focus of ongoing work.

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