Investigating the role of a kinesin gene in butterfly mimicry

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. Here we will continue to
develop genomic resources for Heliconius and reveal the molecular basis for wing pattern specification. Specific goals
are as follows:
1. Generate a high density SNP linkage map using RAD genotyping for scaffolding of the Heliconius melpomene
genome sequence. We will take advantage of modern 'next generation' sequencing technology to generate a high
density linkage map based on short sequence tags. These will form a chromosomal scaffold for assembly of whole
genome shotgun sequence contigs. This will form an important test of feasibility for assembly of a genome using nextgen
sequencing technology.
2. Use sequence capture technology to resequence both genome-wide polymorphic sites and across colour pattern
candidate regions. These data will characterise both genome-wide and local patterns of haplotype diversity and linkage
disequilibrium between six divergent colour pattern races. This will serve to identify putative functional sites that show
fixed differences between races, and provide a comparative analysis of the haplotype structure associated with parallel
divergence in colour pattern across the range of H. melpomene.
3. Characterise splice variants and spatial localization of HmB kinesin gene expression in different phenotypes. We
have already demonstrated spatially restricted expression of the kinesin gene in the distal region of the developing wing,
and will here investigate how this pattern varies between divergent phenotypes of H. melpomene, and whether patterns of
alternative splicing are associated with wing polymorphism. Spatial correlations between gene expression and wing
phenotype will provide a powerful indicator of the mechanism of wing pattern specification between phenotypes.
4. Investigate the molecular function of the kinesin gene through Yeast 2-hybrid screens and test of motor function.
We will investigate which proteins interact with the kinesin molecule, in order to determine how it functions in wing pattern
specification. We will also express the protein in a bacterial system confirm that it is indeed a motor protein with a test of
motility on polarised and immobilised microtubules.
5. Transgenic test of function in Heliconius. We will demonstrate the role of the kinesin gene in wing development
by generating a transgenic butterfly with the red allele at this locus in a yellow banded genetic background. This will
provide the first explicit test of the role of a wing patterning gene in a butterfly.
In summary, we combine a whole-genome analysis of the genetic patterns underlying mimicry evolution with a focussed
investigation of the best candidate yet identified for a wing patterning gene in butterflies.

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
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butterfly

The evolutionary importance of hybridization and introgression has long been debated. Hybrids are usually rare and unfit, but even infrequent hybridization can aid adaptation by transferring beneficial traits between species. Here we use genomic tools to investigate introgression in Heliconius, a rapidly radiating genus of neotropical butterflies widely used in studies of ecology, behaviour, mimicry and speciation. We sequenced the genome of Heliconius melpomene and compared it with other taxa to investigate chromosomal evolution in Lepidoptera and gene flow among multiple Heliconius species and races. Among 12,669 predicted genes, biologically important expansions of families of chemosensory and Hox genes are particularly noteworthy. Chromosomal organization has remained broadly conserved since the Cretaceous period, when butterflies split from the Bombyx (silkmoth) lineage. Using genomic resequencing, we show hybrid exchange of genes between three co-mimics, Heliconius melpomene, Heliconius timareta and Heliconius elevatus, especially at two genomic regions that control mimicry pattern. We infer that closely related Heliconius species exchange protective colour-pattern genes promiscuously, implying that hybridization has an important role in adaptive radiation.
from Dasmahapatra K, Heliconius Genome Consortium (2012) Butterfly genome reveals promiscuous exchange of mimicry adaptations among species. Nature 487: 94-98.