On the mechanistic basis of natural polymorphism in the enzyme G6PD in Colias butterflies [electronic resource]

The study of natural genetic variation has been central to evolutionary biology since its synthesis with genetics in the middle of the last century. In the ensuing time, a handful of case studies have shown how changes at the DNA level influence proteins and their function, organismal phenotypes and performance, and fitness, finding links between each level of biological organization. Patterns and generalizable principles that explain the commonality and diversity of these evolutionary stories remain largely elusive, however. Our lab has sought a systematic approach to exploring the mechanistic basis of natural polymorphism, using central metabolic enzymes in sulphur butterflies of the genus Colias as a model system. One of these enzymes, phosphoglucose isomerase, has been studied in great detail. Another, glucose-6-phosphate dehydrogenase (G6PD), has previously-explored natural variation in both humans and the fly Drosophila melanogaster. Earlier work in our lab showed that G6PD in Colias eurytheme has a single high-frequency amino acid polymorphism in the wild, a glutamic acid (Glu) to glutamine (Gln) mutation in the 57th codon position. This polymorphism is linked to variation in two important fitness components. First, heterozygotic males have greater mating success in the field than do homozygotes, an effect also seen in the sister species Colias eriphyle. Second, females mated to heterozygotic males have greater fecundity than those mated to males of the more common homozygote. My research has sought to connect the observed variation at the nucleic acid and protein levels to the corresponding fitness effects through mechanistic study. First, I examined genetic variation in a related species, Colias meadii, which has previously been shown to harbor the same polymorphism at codon 57, but with a much lower frequency of the minor allele. While this allele did not appear at all in the sequences reported here, another charge-changing amino acid polymorphism undetectable on allozyme gels occurs just five residues away at codon 62. Whether this neighboring variation exhibits any of the same effects on fitness is currently unknown. Second, the study of genetic variation in the G6pd gene was extended to six additional species within the genus, including the aforementioned Colias eriphyle that also exhibits heterozygote advantage in male mating success. The 57 Glu/Gln polymorphism is found in all members of the occasionally hybridizing lowland species complex, which includes Colias eurytheme and Colias eriphyle as well as two other species. This shared high-frequency variant stands in stark contrast to the otherwise remarkably low genetic variation seen in all species for this gene. Third, I assessed genotypic effects on organismal phenotypic traits that might help explain the observed fitness differences. In particular, if males are more persistent in courtship when they have more resources available for mating (the "honest salesman" hypothesis), then differences in male reproductive traits could explain both female fecundity and male mating success effects. Across a suite of mass and biochemical measurements, however, no effect of G6pd genotype was seen. Looking at an earlier life stage, on the other hand, heterozygotes of both sexes appeared to have a small increase in pupal mass compared to homozygotes. Fourth and lastly, I measured in vitro functional properties of the G6PD enzyme as a function of genotype. While there was considerable by-replicate noise in enzyme kinetics data, on average 57 Gln homozygotes displayed a reduction in the catalytic constant and the Michaelis constants for both substrates, as compared to heterozygotes or 57 Glu homozygotes. No thermal stability differences were found between allozymes. While a lot of open questions remain to connect the past and present findings into a complete evolutionary picture, a great deal of progress has been made to fill in some of the gaps. G6PD and PGI, despite being metabolically adjacent, reflect very different evolutionary experiences in the mechanisms that maintain their natural variation.

Keywords: Biology.
Advisor: Stearns Tim primary advisor. Thesis advisor
Watt Ward B.
Herschlag Daniel advisor. Thesis advisor
Petrov Dmitri Alex
Rosenberg Noah