With Flying Colors: Using Drosophila Pigmentation to Study How Differences in Traits Arise

Abstract

Understanding how genotypes influence the production of novel phenotypes and contribute to phenotypic diversity is a fundamental goal in biology. When testing for functional divergence in alleles contributing to phenotypic divergence, researchers often utilize transgenic animals to examine the effects of divergent alleles. Here, I test the commonly held assumption that using of a single, defined genomic location to test for functional divergence between alleles effectively controls for genomic position effects. I find that the relative difference detected between alleles varied across genomic locations, including a single genomic location which allowed expression sufficient to rescue a mutant phenotype, but that failed to detect functional divergence between alleles that was present at other genomic locations. Taking these results into consideration, I used transgenic Drosophila melanogaster flies to investigate the role of noncoding DNA sequences in D. americana and D. novamexicana tan in phenotypic divergence in pigmentation between these species. I found that the 5’ half on tan intron 1 from D. novamexicana in an otherwise D. americana tan allele was sufficient to lighten pigmentation compared to that driven by the D. americana tan allele. The molecular mechanism through which noncoding changes in tan contribute to pigmentation divergence between D. americana and D. novamexicana is investigated. D. melanogaster transgenics expressing the D. americana or D. novamexicana tan transgenes were not an amenable system for drawing convincing conclusions regarding mRNA expression level and the impact noncoding sequence changes have on expression. However, in silico predictions into changes in transcription factor binding sites between D. americana and D. novamexicana revealed three transcription factors with predicted binding sites overlapping a derived sequence change in D. novamexicana. These transcription factors and their binding sites represent candidates for a molecular mechanism through which changes in noncoding sequences in these species could contribute to tan gene expression and/or phenotypic divergence. I created green fluorescent protein (GFP) reporter genes to test for the presence of enhancer sequences in D. americana and D. novamexicana intron 1 and intron 3. All constructs GFP expression in D. melanogaster transgenics, indicating these noncoding sequences have regulatory ability. Finally, I use intraspecific pigmentation variation within D. americana to gain insight into the similarities and differences in phenotypic evolution within and between species. I found that tan and ebony contribute to pigmentation divergence within D. americana for some but not all comparisons, suggesting that additional genes are also involved in the pigmentation variation within D. americana. Finally, by comparing phenotypically similar strains of D. americana, I uncovered evidence supporting the existence of genetic heterogeneity within D. americana. Overall, this research presents important considerations for transgenic analyses using defined genomic integration sites, provides evidence for noncoding DNA sequence in tan contributing to phenotypic evolution between D. americana and D. novamexicana, generates testable hypotheses regarding the molecular mechanism through which noncoding changes in tan contribute to pigmentation divergence between D. americana and D. novamexicana, and offers preliminary data into the genetic loci underlying pigmentation variation within D. americana.