Investigating the role of a kinesin gene in butterfly mimicry

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Biological Sciences

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 collaborations between Dr Jiggins at the University of Cambridge, Prof. ffrench-Constant at the University of Exeter and Prof Blaxter in Edinburgh. 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. The Edinburgh Lab has a proven expertise in genomics, and with recent investemants from MRC (£2.4 M), SULSA and NERC is well placed to deliver on the high throughput sequencing required.

Publications

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Nadeau NJ (2012) Genomic islands of divergence in hybridizing Heliconius butterflies identified by large-scale targeted sequencing. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

 
Title Transmissions exhibition 
Description The Blaxter lab collaborated closely wth artists-in-residence (see http://www.ascus.org.uk/ciie-micro-residency-artists-announced/) in the Centre for immunity Infection and Evolution to inspire and be part of the final exhibition "Transmissions". Mark Blaxter appears in the film work produced by Anne Milne, and the work of the lab inspired Jo hodges and Robbie Coleman to produce a piece dedicated to the lab. 
Type Of Art Artwork 
Year Produced 2014 
Impact 'Transmissions' was showcased to the general public within a group exhibition 'Parallel Perspectives' in Summerhall as part of the Edinburgh International Science Festival 2015 art programme, How The Light Gets In . This exhibition of work susequently travelled LifeSpace, Dundee, returning to Edinburgh to showcase at the Tent Gallery, Edinburgh College of Art. 
URL http://www.ascus.org.uk/ciie-micro-residency-2/
 
Description We have identified a novel mechanism whereby animals can become more diverse - by sharing key sets of genes between species (in our case, different butterfly species have shared genes in the distant past that affect important colour patterns on their wings).
Exploitation Route The idea that species might continue to share some genes after they have split from their ancestors - breaking down, even if temporarily, species barriers, may have implications for conservation of endangered taxa.
Sectors Environment

 
Description BBSRC Project Grant (Genome Databasing)
Amount £671,655 (GBP)
Funding ID BB/K020161/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2013 
End 11/2016
 
Title LepBase - lepidoptera genome database 
Description LepBase is a tier two ENSEMBL database for the genomes of all lepidoptera. Genome sequencing, and large-scale, population genomic analysis, has suddenly become affordable. The explosion of data presents tremendous opportunity for ground-breaking research based on integration of data from independently-organized, community-driven genome projects, but this in turn requires shared database resources. For model organisms, genome databasing efforts grew with the research communities, and there are mature portals for deep investigation across many large scale datasets - the databases themselves have become a substrate for (meta-) research of high impact. For communities new to genomic (and population genomic) approaches, the need for accessible databases is even more pressing, as researchers are less likely to be fluent in the peculiar languages of genomics and in high-throughput bioinformatics. Here we propose the founding of a community database for lepidopteran genomics, LepBase, to meet the needs of the growing community of researchers using genomics to understand Lepidoptera as crop pests, as potentially invasive species, as developmental models, and as key taxa for understanding the interplay between ecology, genomics, evolution and speciation. While initially focussed on the available lepidopteran genomes the project will meet the challenge of future genomic riches (over 20 genomes 'in the pipeline') by building a platform that focuses on the needs of the lepidopteran research community. The challenge of integrating newly developed genomic resources across taxa is not a new one, and several computational frameworks exist to support such endeavors (such as the ENSEMBL project, and the GMOD ecosystem of tools). Central, aggregative database efforts, such as ENSEMBL Genomes, provide an effective and powerful, one-size-fits-all solution to genome warehousing. Coordinating with smaller research communities to directly implement clade-specific resources is overwhelming the resources of institutions that have a mandate to generate integrated genomic databases. ENSEMBL now advocates a multi-tiered approach to the aggregation, integration, and dissemination of the rapidly increasing wealth of genomic information arising from community-driven genome projects so that species-level genomic resources can flow 'upstream' into the pan-genome database. The goals of our project are: to develop a community-wide, comparative database for the Lepidoptera using the ENSEMBL platform; to institute effective tools for ongoing community annotation of emerging genomes; to forge close links with ENSEMBL Genomes to ensure upload of lepidopteran genomes into the global resource; to implement new modes of data visualisation and analysis in the ENSEMBL framework to meet community needs; and to provide training in genomics to the community of lepidopteran researchers. The LepBase database will also be a working model of community-driven databases that drive not only clade-specific research programmes but also enable the flow of knowledge from species-specific genome projects into a comprehensive framework. The project will be based in the Blaxter bioinformatics and genomics group in Edinburgh, in association with the GenePool Genomics Facility (currently engaged in sequencing butterfly and moth species), with project partners in the Jiggins Heliconius research group in Cambridge and Dasmahapatra in York, and the support of lepidopteran researchers worldwide. Initial focus will be on the genus Heliconius, for which a complete genome sequence and abundant annotation, transcriptome and resequencing data already exist. The database will be rapidly extended into silkmoth, Bicyclus, Danaus and other species. The resource will be overseen by a Scientific Advisory Board drawn from across the range of lepidopteran researchers, and will aim for financial sustainability beyond the tenure of the award through development of a 'subscription' model of funding from research partners. 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact N/A 
URL http://lepbase.org/
 
Description Heliconius genome consortium 
Organisation University of Cambridge
Department Department of Zoology
Country United Kingdom 
Sector Academic/University 
PI Contribution Offering genomic sequencing and reseaquencing technologies; close involvement in experimental design.
Collaborator Contribution Joining the consortium has allowed GenePool to develop custom targeted resequencing technologies, and associated bioinformatics skiils, and made us much more visible across this area of science
Impact The Heliconius melpomene genome has been assembled and annotated. A manuscript describing this work is submitted for publication. On the basis of the GenePool's involvement in this consortium we have coordinated a ladybird genome consortium with 6 partner laboratories, and currently have 2 genomes in sequencing.
Start Year 2010
 
Title EasyMirror/EasyImport 
Description These tools simplify the rool out of customised ENSEMBL databases. 
Type Of Technology Software 
Year Produced 2016 
Open Source License? Yes  
Impact We have been inundated with requests for support and assistance in delivering these tools, and with praise for their ease of use and timeliness. 
URL http://www.genomehubs.org
 
Description Blaxter group - presentations and outreach 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact The Blaxter group presented work at a wide range of national and international conferences, including PopGroup, the Arthropod Genomics Workshop, The C. elegans International Meeting, The Hydra Helminthology meeting, The European Society for Nematology, The UK Genome Science meeting, and others. At many of these venues, in addition to offering platform or poster presentations, we also presented workshops or training activities.
Year(s) Of Engagement Activity 2016
 
Description Blaxter group presentations and outreach 2015 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Mark Blaxter and research team communication and outreach 2015

Globodera genomics and blobtools software
25/02/2015 JHI Postgraduate Student Competition 2015 James Hutton Institute, Aberdeen, UK A tale of Two Peaks: Analysing Genomic Data from Potato Cyst Nematodes Talk
26/03/2015 JHI Cell and Molecular Sciences (CMS) seminar James Hutton Institute, Invergowrie, Dundee, UK Frustration and happiness : (De)-constructing parasite genomes Talk
16/06/2015 JHI Dundee effector consortium (DEC) meeting 2015 Birnam Arts and Conference Centre, Birnam, UK Variation within the Globodera pallida species complex: preliminary results Talk
03/09/2015 Molecular and Cellular Biology of Helminth Parasites IX Bratsera Hotel, Hydra, Greece Inter- and intra-specific analyses of the effector complement in potato cyst nematodes Poster
18/09/2015 UoE Postgraduate Poster Day University of Edinburgh, Edinburgh, UK Inter- and intra-specific analyses of the effector complement in potato cyst nematodes Poster
26/09/2015 Edinburgh University Doors Open Day University of Edinburgh, Edinburgh, UK Potato Cyst Nematodes (PCN) - Nematode parasites of potatoes Poster
30/11/2015 NextGenBug University of Edinburgh, Edinburgh, UK Blobtools: Blobology 2.0 Talk
01/12/2015 UK pollinator genomics meeting Roslin Institute, Edinburgh, UK Bees and Blobs Talk

LepBase
06/03/2015 EMARES Cambridge, UK The Bicyclus Genome Project Talk
06/03/2015 EMARES Cambridge, UK An introduction to Lepbase Talk
17/06/2015 Arthropod Genomics Manhattan, Kansas, USA Lepbase - A multi genome database for the Lepidoptera Poster
24/07/2015 10th Heliconius Meeting Gamboa, Panama Lepbase - A multi genome database for the Lepidoptera (API demonstration) Workshop
24/07/2015 10th Heliconius Meeting Panama Lepbase - A multi genome database for the Lepidoptera Poster
26/07/2015 10th Heliconius Meeting Panama Lepbase Workshop Talk
04/09/2015 Edinburgh Bioinformatics Edinburgh, UK Lepbase - A multi genome database for the Lepidoptera Talk
26/09/2015 Open Doors Day "Make a butterfly" interactive exhibition
26/09/2015 Edinburgh University Doors Open Day Edinburgh, UK Lepbase Multiple Sequence Alignments game Poster+Game
28/10/2015 NextgenBUG Dundee, UK Lepbase - an Ensembl (and more) for the Lepidoptera Talk

Nematode genomics
24.06.2015 20th International C. elegans Meeting Los Angeles USA A new evolutionary framework for the phylum Nematoda: a case study of HOX cluster evolution Poster
24.06.2015 20th International C. elegans Meeting Los Angeles USA Caenorhabditis Genomes Project Workshop (organiser and chair) Talk
24.06.2015 20th International C. elegans Meeting Los Angeles USA Current status of the CGP in Edinburgh Talk

Meloidogyne genomics
10-14 August 2015 ESEB Lausanne-Switzerland Genomic consequences of hybridization and the loss of meiotic recombination in Root-knot nematodes poster
15-18 December 2015 PopGroup Edinburgh-UK Genomic consequences of hybridization and the loss of meiotic recombination in Root-knot nematodes talk
23 February 2016 NextGenBug Edinburgh-UK Genomics of Root-knot nematodes talk
Year(s) Of Engagement Activity 2015
 
Description Blaxter lab workshops 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The Blaxter lab took our software products and research tools to various venues (Arthropod Genomics, UK Genome Science meeting, Butterfly Genomics) to present as workshops, training events or interactive sessions
Year(s) Of Engagement Activity 2016
 
Description Press releases and website 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact We have engaged actively with the University press office to promote press coverage of our research outcomes, particularly major publications (which have had coverage in national and international newspapers) and in blogs and other online media. We have also promoted major new initiatives such as additional core funding of the Edinburgh genomics facility.

Increased visibility of Edinburgh Genomics within the community; requests for comment by funders and government on matters pertaining to genomics.
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014,2015,2016