Investigating the role of a kinesin gene in butterfly mimicry

Lead Research Organisation: University of Cambridge
Department Name: Zoology

Abstract

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.

Technical Summary

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.

Planned Impact

Ever since Bates, Darwin and Wallace described mimicry in Amazonian butterflies, scientists have been interested in understanding its genetic control and evolutionary origins. Biology textbook still describe mimicry as a central paradigm of Natural Selection. However despite the level of interest, the gene we investigate here is arguably the first candidate gene for the evolutionary diversification of butterfly wing patterns. The impact of this grant is therefore in the publication and public interest in butterfly mimicry. 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 itself and the techniques we are using are of interest to the UK public. 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 publications ranging from the Guardian, Times, El Globo (Brazil), to the West Briton, we have achieved substantial media coverage. Web based data-bases and tool development: As well as technique development our work on butterflies has led to the creation of a unique database 'ButterflyBase' for the curation and analysis of non-Drosophilid insect sequences. 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: We have developed the wider outreach of our work through our Heliconius web site, which provides information on the biology of the butterflies in an accessible format and allows for the community to post articles of general interest. The site has received over 6000 visitors in the six months since launch of the updated site in January 2009. This project will offer an opportunity to take advantage of this popularity to promote genomics as well as in teaching modern evolutionary biology. In addition, the applicants will also maintain personal websites with updates on research, recent publications and press releases and 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 and training: 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 £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. We will use the new posts gathered to drive further work in butterflies and natural selection at Exeter and in continued collaboration with Cambridge. The Jiggins group has also played an important role in developing educational links with Latin America, by hosting visiting students from Colombia, recruiting latin American PhD students and providing opportunities for research interns in our facility in Panama. Over the years, students with whom we have worked have gone on to do PhDs in the UK and US and successful research careers.

Publications

10 25 50
 
Description Mimicry among butterfly species is a commonly cited example of evolution and adaptation and Heliconius are perhaps the best studied example, but the molecular genetic basis of mimicry has only recently been studied. The goals of this project were to continue to develop genomic resources for Heliconius and reveal the molecular basis for wing pattern specification at the BD locus.

We have published a RAD sequence linkage map that was used to scaffold 85% of the H. melpomene genome. This work was fundamental for our recent publication in Nature of the H. melpomene genome sequence. We are now working on version 2 of the genome assembly, and improving the genome is an ongoing project, but the published work demonstrates that we have already achieved this goal as planned

We have obtained both SureSelect sequence capture data and whole genome sequences for ~80 individuals across the BD locus. This has provided remarkable resolution towards isolating associated sites across the BD locus, and permits separation of loci associated with distinct patterning elements (see Fig. 1). This has shown that shared patterns in the melpomene group have arisen through adaptive introgression (2,3) and that population divergence in centred on non-coding sequence between optix and kinesin (1). In particular we can separate the red basal forewing patch (Dennis) and the hindwing ray pattern elements (Rays) and localise associated haplotypes to regions of 6kb and 12kb respectively.

We have used both qPCR and in situ hybridisation to show that the kinesin gene is expressed in association with the red forewing band in both H. melpomene and H. erato. This confirms parallel and independent co-option of this gene into wing patterning in these convergent species. However, kinesin is not associated with any other pattern elements tested so far, including the rayed pattern, which is also controlled by this BD locus. So far we have not demonstrated any consistent slice variants associated with patterning, so it is unclear whether alternative splicing is going to be an important mechanism. In collaboration with Dr Reed, we have shown that expression of the transcription factor optix is associated with all red patterning elements (Reed et al., 2011). Furthermore, Since both kinesin and optix are associated with pattern, we have been cautious about proceeding with more complex functional experiments on the kinesin gene. Instead we have carried out an additional microarray expression experiment to test patterns of differential expression both between wing regions and between races. This has clearly supported the role of the kinesin in pattern specification alongside optix. Kinesin is expressed earlier in development as compared to optix (Figure 1), supporting a functional role for both genes.

Finally published in 2016 in PLoS Biology, we have dissected the genetic basis for distinct phenotypic elements in the red locus, showing that different haplotypes have very different evolutionary histories. This provides a textbook case of adaptive introgression between species.
Exploitation Route Further research into patterning evolution in butterflies and other organisms.
Sectors Environment

URL http://www.heliconius.org
 
Description We have used the results of this work in our presentation for the Royal Society Summer Science Exhibition 2014 in which we described the evolution of butterly wing patterns to ~15000 members of the public
First Year Of Impact 2014
Sector Education,Environment
Impact Types Societal

 
Description BBSRC BBR Fund
Amount £534,491 (GBP)
Funding ID BB/K020161/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2013 
End 06/2017
 
Description ERC Advanced Grant
Amount € 2,500,000 (EUR)
Funding ID SpeciationGenetics 
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Start 06/2014 
End 06/2019
 
Description Research Grant
Amount £245,000 (GBP)
Funding ID RPG-2014-167 
Organisation The Leverhulme Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2014 
End 09/2017
 
Description Seeding Catalyst Award
Amount £18,500 (GBP)
Funding ID BB/SCA/Cambridge/17 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 03/2019
 
Title Advances in use of ABBA-BABA statistics for detecting introgression 
Description This is an advance in the analytical methods used to detect introgressed genome segments in genomic data 
Type Of Material Improvements to research infrastructure 
Year Produced 2014 
Provided To Others? Yes  
Impact The methods were published in 2014 in this paper and have been used quite widely by other research groups Mol Biol Evol (2014) 32 (1): 244-257. 
URL https://doi.org/10.1093/molbev/msu269
 
Title LepBase 
Description LepBase is a community resource providing lepidopteran genome sequences in a genome browser based on the ENSEMBL format. This provides tools for comparative genome analysis. This database is the first community level database to use the ENSEMBL genome browser established outside the EBI and as such provides a model for future community genomics databases 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact The resource is being widely used by the lepidopteran genome community for functional and research applications 
URL http://ensembl.lepbase.org/index.html
 
Description Heliconius Genome Consortium 
Organisation Baylor College of Medicine
Department Human Genome Sequencing Centre
Country United States 
Sector Academic/University 
PI Contribution I led establishment of the Heliconius Genome Consortium which sequenced the first butterfly genome
Collaborator Contribution Many skills in genomic analysis and Heliconius biology
Impact Genome sequenced and published
Start Year 2010
 
Description Heliconius Genome Consortium 
Organisation Cornell University
Country United States 
Sector Academic/University 
PI Contribution I led establishment of the Heliconius Genome Consortium which sequenced the first butterfly genome
Collaborator Contribution Many skills in genomic analysis and Heliconius biology
Impact Genome sequenced and published
Start Year 2010
 
Description Heliconius Genome Consortium 
Organisation Harvard University
Country United States 
Sector Academic/University 
PI Contribution I led establishment of the Heliconius Genome Consortium which sequenced the first butterfly genome
Collaborator Contribution Many skills in genomic analysis and Heliconius biology
Impact Genome sequenced and published
Start Year 2010
 
Description Heliconius Genome Consortium 
Organisation National Museum of Natural History
Country United States 
Sector Academic/University 
PI Contribution I led establishment of the Heliconius Genome Consortium which sequenced the first butterfly genome
Collaborator Contribution Many skills in genomic analysis and Heliconius biology
Impact Genome sequenced and published
Start Year 2010
 
Description Heliconius Genome Consortium 
Organisation Smithsonian Institution
Department Smithsonian Tropical Research Institute
Country Panama 
Sector Academic/University 
PI Contribution I led establishment of the Heliconius Genome Consortium which sequenced the first butterfly genome
Collaborator Contribution Many skills in genomic analysis and Heliconius biology
Impact Genome sequenced and published
Start Year 2010
 
Description Heliconius Genome Consortium 
Organisation University of the Andes, Chile
Country Chile 
Sector Academic/University 
PI Contribution I led establishment of the Heliconius Genome Consortium which sequenced the first butterfly genome
Collaborator Contribution Many skills in genomic analysis and Heliconius biology
Impact Genome sequenced and published
Start Year 2010
 
Description Royal Society Summer Science Exhibit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact 15000 members of the public and schoolchildren attended the Royal Society Summer Science Exhibit in London and learnt about our work on wing pattern evolution in Heliconius

Feedback from teachers and schoolchildren indicated that we inspired new ways of thinking and understanding genetics and evolution
Year(s) Of Engagement Activity 2014
URL http://sse.royalsociety.org/2014/butterfly-evolution/