The molecular causes of convergent evolution

1) The big questions
Convergent evolution is a natural experiment in repeated evolution of similar phenotypes, offering unique insights into the evolutionary process. When similar patterns evolve in different lineages, to what extent are the same molecular mechanisms deployed? Are the same regulatory changes co-opted into generating convergent phenotypes? How can evolutionary change at a single locus regulate complex patterning changes during rapid evolution? What are the precise genetic changes necessary for the evolution of a new developmental pattern? Heliconius butterflies are an excellent system to address these questions.

2) The background
Many tropical butterflies have mimetic wing patterns to warn predators of their toxicity, and these have become an excellent system in which to understand the molecular basis for convergence and diversification, and make the link between natural selection in the wild and evolution in the genome. Here we will study the molecular basis for pattern convergence in tropical Heliconius butterflies. Genetic mapping and gene expression experiments have identified a simple system of three genetic loci that control the complex diversification in wing patterning seen in Heliconius. One of these loci regulates yellow pattern elements and the same genomic locus is also involved in wing patterning of both the peppered moth, Biston betularia, and the butterfly Bicyclus anynana, suggesting an ancient shared patterning system in butterflies and moths. Patterns of expression and genetic data from natural populations, suggest that cortex is the functional gene at this locus, although expression data also point to involvement of other linked genes. We will apply recently developed CRISPR/Cas9 methods for gene knockouts to investigate the molecular basis for pattern convergence between species of Heliconius butterflies.

3) Objectives and expected results
We will test the frequency with which similar patterns evolving in mimetic butterflies use the same genes. At a closer resolution, we will also test whether those genes are controlled by the same regulatory switches to turn them on and off, when they control similar patterns. This will test for the repeatability of evolution in different genetic backgrounds. These experiments will involve developing and applying novel gene editing techniques to study patterning in these butterflies, which will set the standard for evolutionary studies in the future. The remarkable patterns of mimicry in these butterflies have long been considered an exemplar of evolution by natural selection, and this project will offer unique new insights into the molecular mechanisms that produce such strikingly similar patterns in so many different species.

Convergent evolution offers a powerful system for testing the repeatability of genetic changes underlying evolution. The wing patterns of Heliconius butterflies are phenotypes subject to strong natural and sexual selection that show convergence due to mimicry as well as great diversity. Here we study on a locus controlling Heliconius patterns that is also involved in wing patterning in another butterfly (Bicyclus anynana) and the peppered moth (Biston betularia). This locus includes the gene cortex, a member of a family of cell cycle regulators, but also two other candidates domeless and washout. We will use CRISPR/Cas9 to induce targeted mutations in these genes among convergent mimetic species, to test the hypothesis that the same genes are involved in generating convergent phenotypes. We will characterise spatial and temporal patterns of expression during development, to test whether the genes are deployed in the same manner to produce convergent patterns. We will then identify candidate regulatory sequence controlling specific pattern elements by combining 1) population association data from genome scale sequence data to identify genetic variants associated with particular phenotypes, and 2) ATAC-seq data to identify open chromatin. The necessity of these regulatory loci in producing convergent phenotypes will be tested using CRISPR/Cas9 directed deletions of candidate enhancer regions. Mapping and functional testing of regulatory loci across convergent mimetic species will for the first time identify the precise nature of regulatory convergence in enhancer elements across an adaptive radiation. Finally, we will conduct reciprocal transfers of regulatory sequence between butterflies to test the sufficiency of these loci in producing convergent phenotypes. This will provide the most detailed genetic analysis to date on the molecular basis for convergent evolution, answering fundamental questions in evolutionary and developmental biology.

Butterflies are charismatic organisms that have great resonance with the British public. We have experience in communicating our work to a wide variety of age groups through the Royal Society Summer Science Exhibition and at Cambridge Science Week, and visits to local primary schools. Here we will work with the Museum of Zoology in Cambridge to develop resources that take advantage of the recent inclusion of evolution as a topic on the KS2 primary school science curriculum. First, we will develop a teaching session for Year 6 primary school children, based around an interactive mimicry and predation game that we have already developed. The session will be developed by the outreach team at the Cambridge Museum of Zoology in collaboration with the PI and PDRA. We will focus on the National Curriculum topics of heredity and adaptation, and use our own research on butterflies to illustrate these topics. This will be delivered by Museum staff to primary school children visiting the museum for school visits during the course of the grant and into the future. Local primary school teachers have indicated a strong demand for help in teaching evolution, as it is not a topic that they have prior experience teaching and many lack formal science training. This will therefore fulfil a considerable local demand. In addition, we will develop an accompanying online package that will allow teachers to conduct follow-on sessions in their own schools and that can also reach a wider audience across the country.

Second, we will organise regular events for state school sixth form students to enthuse them about biology and expose them to primary research. The goal is to encourage an interest in cutting-edge scientific research and widen the diversity of undergraduate applicants to study biology at university. These events will be hosted at St John's College in Cambridge and will involve the top students from state secondary schools at a national level. We will also continue to use the Heliconius.org web site to advertise our work and communicate to our regular followers, and release regular press releases and web articles about our findings as they are published.