Investigating the role of a kinesin gene in butterfly mimicry

Mimicry among butterfly species is a classic example of evolution and adaptation. The brightly coloured neotropical Heliconius butterflies are one of the best studied examples, but the molecular genetic basis of mimicry remains poorly understood. In particular mimicry offers an opportunity to study the repeatability of evolution, as the same patterns emerge again and again in divergent lineages. The project falls into two broad areas, first we use cutting edge sequencing technology to make a genetic map of the H. melpomene genome. This will be used to help assemble the genome sequence currently being generated. This will form the basis for a genome-wide survey of adaptive divergence between H. melpomene races. Divergent geographic populations of this species form narrow hybrid zones where they hybridise and exchange genes. Thus, narrow regions of the genome controlling wing patterns are genetically differentiated against a background of extensive recombination. This offers a powerful opportunity to identify changes responsible for wing pattern differentiation. We will first characterise variation within and between races by genome resequencing at low coverage. Then we will use novel 'sequence capture' technology to enrich genomic DNA for regions of interest from 96 individuals, taken from six phenotypic races of H. melpomene. The experiment will be designed to sample two chromosomal regions containing wing patterning genes, and a further 12,000 variable sites located across the genome. This will offer a unique genome-wide analysis of parallel divergence between the six populations sampled. In particular we aim to determine a) how much of the genome is involved in colour pattern divergence b) whether the same regions are implicated across independent hybrid zones c) estimate the age of the alleles involved in wing pattern divergence and d) identify putative functional sites for further analysis. The second major aim of the project is to investigate the kinesin gene that represents a strong candidate locus for controlling the red forewing band of H. melpomene. We will study the spatial distribution of kinesin gene expression patterns between divergent phenotypes, in order to test whether spatial regulation underlies pattern regulation. Many genes show different variants generated by alternative splicing, generating variant forms of the protein containing alternative forms of the exons. Alternative splicing is a potentially powerful but under-explored mechanism that could generate evolutionarily relevant variation. We have evidence for alternative splicing the Kinesin gene, and here will characterise the isoforms of this gene and test for correlations between isoform expression and wing phenotype. We will investigate the molecular function of the gene, including a search for other molecules that interact with the kinesin protein, and test to confirm its motor function. Finally, we will develop trangenics methods for explicitly testing the function of the kinesin gene in wing pattern specification in divergent races of H. melpomene. The major gene dominant control of the red band means that we expect to be able to generate a red-banded phenotype by expressing the 'red' kinesin allele in a yellow banded phenotype. This will provide the first explicit test of function for a gene causing pattern variation in any butterfly.

Mimicry among butterfly species is a commonly cited example of evolution, and Heliconius are perhaps the best studied case, but the molecular genetic basis of mimicry remains poorly understood. Mimicry offers an opportunity to study the repeatability of evolution, as the same patterns emerge again and again in divergent lineages. The project falls into two areas, first we will carry out a genome scale population survey of adaptive divergence. First, a linkage map will be constructed using RAD tag Illumina sequencing to scaffold genome sequence contigs being generated by Baylor HGSC. This will improve assembly of the genome sequence currently in production. Then we will generate low coverage genome re-sequencing of six races of H. melpomene to identify variable sites. These will be used to design a sequence capture experiment to sample both 1.8MB across two wing pattern candidate regions, and 12,000 SNPs located across the genome. This will offer a unique genome-wide analysis of parallel divergence across three wing pattern hybrid zones. The second major aim of the project is to investigate the kinesin gene that represents a strong candidate locus for controlling the red forewing band of H. melpomene. We will study how spatial expression patterns of the gene vary between divergent phenotypes using in situ hybridisation, to test whether spatial regulation underlies pattern regulation. We will characterise the isoforms of this gene and test for correlations between isoform expression and wing phenotype. We will investigate the molecular function of the gene, including a search for interactor molecules and test of motor function. Finally, we will carry out a transgenic test of function of the kinesin gene by inserting a construct of 'red' allele of kinesin plus potential promotor sites into a 'yellow-banded' phenotype. This will be the first functional investigation of a gene underlying the spectacular adaptive radiation of butterflies.

Ever since Bates, Darwin and Wallace themselves described mimicry in Amazonian butterflies such as Heliconius, scientists have been interested in understanding its genetic control. Thus if you open any basic textbook of Biology you will find mimicry quoted as a central paradigm of Natural Selection. However, despite the level of interest in mimicry it is only in this grant that we investigate a candidate gene for mimicry in Heliconius for the very first time. The main impact of this grant is therefore in the publication and public interest within butterfly mimicry itself. Our previous grant in this subject area was heavily quoted by the press and we anticipate similar coverage for this proposal. Initially it is hard to see how investigation of a kinesin (a cell motor protein) in butterfly wing colour could be of anything but 'academic' interest. However our previous experience suggests that both the gene is of interest to the UK public and also the techniques we are using to investigate the gene. Press releases: We have run joint press releases with the University of Cambridge and University of Exeter press offices to cover work on butterfly wing colour. Variously described as 'Painting by numbers' and 'Natural selection takes flight' in articles ranging from the Times to the West Briton, we have achieved substantial media coverage and interest. Techniques and their application to UK science: Several of the techniques we are using in this proposal are 'cutting edge' and key to UK science remaining competitive with the USA. For example the 'pull-down' sequencing we are doing with the Liverpool Unit of Genomics has not been done on non-model insects before and its use and development at Liverpool is likely to add value to UK bioscience. Further, our use of RAD tagging will also be useful in the development of linkage maps for mapping in non-model genomes. Web based data-bases and tool development: As well as technique development this work on butterflies has led to the creation of a unique data base 'ButterflyBase' for the curation and analysis of non-Drosophilid insect sequences. At ButterflyBase you can not only locally BLAST search (ICEBlast) collections of insect ESTs but you can also view sections of the butterfly genome and compare them between species to look for synteny. The development of such databases will not only be useful for butterflies but will also serve as a repository for all other non-Drosophilid insect sequences. Communication and engagement: The applicants will not only maintain their personal websites with updates on their research, recent publications and press releases but they will also actively campaign to publicly disseminate the information. Both Dr Jiggins and Professor ffrench-Constant are excellent public speakers who are used to addressing non-academic audiences in venues such as 'Café Scientifique' and addresses to the Royal Society (Dr Jiggins is a URF and Prof. ffrench-Constant a past Merit Award Holder). Collaboration: This proposal results from a long ongoing collaboration between Dr Jiggins at the University of Cambridge and Prof. ffrench-Constant at the University of Exeter. The work at each university has been used as a flag-ship to attract further funds from the Leverhulme Trust for Dr. Jiggins and also £30 million for a new institute of Environmental Sustainability at Falmouth with 10 new posts with Prof. ffrench-Constant as Chair of the Search Committee. Added value and the Environmental Sustainability Institute (ESI) at Falmouth: The butterfly work at Falmouth and other work in the environment has been key in the university being awarded 30M by the RDA and the EU. We will use the 10 posts gathered to drive further work in butterflies and natural selection at Exeter and in continued collaboration with Cambridge. The first ESI post has already been appointed, Dr Chris Wheat from Hanski's group and we will hire two new posts per year for each of the next 5