We seek to understand the evolution of morphological novelties by focusing on the evolution and development of butterfly wing patterns. Research in the lab addresses both the ultimate selective factors that favor particular wing patterns, as well as the proximate mechanisms that generate those patterns. We combine tools from ethology, population genetics, phylogenetics, and developmental biology to understand the nature of the variation underlying developmental mechanisms within or between species, and why species display their particular color patterns. Our model organisms (so far) have been African satyrid butterflies in the genus Bicyclus, pierid butterflies, and saturniid moths.
If you are interested in pursuing a graduate project (PhD project in the lab) you will have to apply to the Department of Ecology and Evolutionary Biology (EEB) at Yale University to enter the graduate program.
If you are an undergraduate, there are several ways that you can participate in research in the lab. Either by doing an honors project (two semester's worth of research), an independent project (one semester), or a summer project (three months).
In order to test whether a gene has a role in the development of butterfly wing patterns it is important to be able to manipulate its expression using transgenic tools. We have successfuly transformed the genome of Bicyclus anynana with Hermes and piggyBac transposable elements. We initially used EGFP and DsRed under the control of different promoters (actin and 3xP3) as marker genes to test germ line transformation. Later we tested whether the heat-shock promoter from Drosophila Hsp70 could be used as an inducible promoter. More recently we have tested the role of several developmental genes in eyespot development by either over-expressing or down-regulating these genes at particular times during development using the Hsp70 promoter. People that did this work were formal post-doctoral researcher Jeffrey Marcus, graduate student Diane Smith, former visiting professor Bin Chen, and myself.
Currently, postdoctoral fellow Andrew Stoehr is also producing transgenic Pieris rapae butterflies using piggyBac constructs.
New over-expression and RNAi plasmid construct:
Bin Chen produced a new expression plasmid that can be used for either gene over-expression (or ectopic expression once activated in a new location) or gene knock-down. This plasmid uses piggyBac as the transposable element, and EGFP under the control of 3xP3 as the transformation marker. Candidate genes, inserted once, can be over-expressed. When inserted twice, in opposite orientation, can start the process of RNA interference (RNAi) inside the cells.
Firdous Kamal developed a new laser-mediated heat-shocking mechanism using a IR laser that enables us to heat-shock specific areas of a transgenic butterfly in order to induce ectopic expression of candidade wing patterning genes. Bin Chen and Diane Smith cloned a series of candidate genes and introduced them in the germ line of B. anynana. These transgenes were under the control of the heat-shock promoter. These experiments will allow us to test the role of these candidate genes in the differentiation of colored wing scales.
Reconstructing and animating ancestral wing patterns:
Undergraduate student, Sam Arbesman, worked on a web based animation (Ancient Wings) that reconstructs the putative ancestral ventral hindwing patterns of 54 of the 80 species of Bicyclus butterflies, and morphs these patterns across the phylogenetic tree of Bicyclus.
Jeff Oliver also reconstructed presense and absence of eyespots across the Bicyclus phylogeny and estimated rates of eyespot evolution separately for males and females. He found that eyespots on the dorsal surface or forewing surfaces were more likely evolving via sexual selection because rates of evolution were high and different between the sexes. Eyespots on the exposed ventral or hindwing surfaces were evolving slowly and at similar rates across the sexes. Jeff's ongoing project extends this work to use wing pattern data to all nymphalid butterflies (using the extensive collection of Lepidoptera housed at the Peabody museum). Essentially we are interested in testing whether the ancestral nymphalid wing pattern is similar to the proposed "Nymphalid Ground Plan". Did eyespot number evolve by the addition of eyespots to more and more wing compartments throughout evolution, or did eyespots appear in all wing compartments originally and were later lost from particular wing compartments?
Testing the role of sexual selection in maintaining species (and season) specific wing patterns:
Graduate students Kendra Roberston and Katie Costanzo discovered that wet season female B. anynana choose males on the basis of their UV-reflective dorsal eyespot pupils as well as their pheromones. When one or both of these two traits are blocked or removed, females don't like to mate with these males. Postdoctoral researcher Katy Prudic discovered that dry season males also care about a female's UV-reflective dorsal eyespot pupils. Females display their dorsal eyespots to males during the dry season, but not during the wet season. Males do the opposite. Females are the choosy sex in the wet season and males are the choosy sex during the dry season. This phenotypically plastic courtship reversal is controlled by larval rearing temperature. Females court males in the dry season because the spermatophore they receive from these males allows them to live almost twice as long as females that don't mate or mate with a wet season male. It is still unclear why males are reticent to mate with dry season females.
Testing the role of natural selection in maintaining species (and season) specific wing patterns:
Katy Prudic is testing the role of mantid predators is explaining the plasticity in eyespot size on the ventral surface of the wings of B. anynana in response to the wet and dry seasons. Vertebrate predators such as birds and lizards appear to have a more difficult time detecting the dry season form relative to the wet season form in cage experiments in the lab. But, once they detect the butterflies, attacks are usually directed towards the wing margin in both forms. So, it is unclear why ventral eyespots become large in the wet season form, given that in interactions with these predators they don't appear to have any advantage. Katy is testing the idea that large eyespot size may have evolved to deflect attacks from insect predators such as mantids that have a different visual system (cannot detect UV). These predators appear to attack the wing margin of butterfies with large contrasting rings of color, but attack more vulnerable parts of the insect if eyespots are small.
Transposable element mediated mutagenesis and enhancer trapping:
This project was started together with former postdoctoral fellow Jeffrey Marcus, but needs further development. The aim is to develop several lines of B. anynana carrying specific constructs. One line will have a stable transposase, other lines will carry hopping elements. We will be crossing these lines in order to induce the transposable elements to hop in the genome of B. anynana. These hopping events may cause interruptions in the sequence of a gene and lead to a visible mutation. Alternatively, the hopping element can integrate close to a gene enhancer region and "trap" that enhancer, i.e., become expressed in the same pattern as the gene that is being enhanced. In both cases, the genomic regions surrounding the hopping element can be readily sequenced and identified.