The molecular causes of convergent evolution

Lead Research Organisation: University of Cambridge
Department Name: Zoology


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.

Technical Summary

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.

Planned Impact

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 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.


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Description This project aims to study convergent evolution in the genes that control wing patterns. We have generated CRISPR mutants for the cortex gene that was a primary goal of the project. We have published this work in Elife, and have demonstrated functional evidence for the role of Cortex and have antibody data shedding light on likely functional mechanisms. We also demonstrated regulatory mutations at the Cortex gene that have localised effects on phenotype that are consistent with known genetics of this locus. This provides support for the modularity of action of regulatory change at Cortex.
Exploitation Route There are still outstanding questions about the mechanism of action of this cell cycle regulator gene in controlling pattern phenotypes.
Sectors Education,Environment

Description The work has been used in presentations to schoolchildren interested in apply to study biology in Cambridge. The work was also used in demonstrating gene editing methods to students on the University of Cambridge Tropical Biology field course in Panama.
First Year Of Impact 2018
Sector Education
Impact Types Societal

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 Cortex cis-regulatory switches establish scale colour identity and pattern diversity in Heliconius 
Description In Heliconius butterflies, wing pattern diversity is controlled by a few genes of large effect that regulate colour pattern switches between morphs and species across a large mimetic radiation. One of these genes, cortex, has been repeatedly associated with colour pattern evolution in butterflies. Here we carried out CRISPR knock-outs in multiple Heliconius species and show that cortex is a major determinant of scale cell identity. Chromatin accessibility profiling and introgression scans identified cis-regulatory regions associated with discrete phenotypic switches. CRISPR perturbation of these regions in black hindwing genotypes recreated a yellow bar, revealing their spatially limited activity. In the H. melpomene/timareta lineage, the candidate CRE from yellow-barred phenotype morphs is interrupted by a transposable element, suggesting that cis-regulatory structural variation underlies these mimetic adaptations. Our work shows that cortex functionally controls scale colour fate and that its cis-regulatory regions control a phenotypic switch in a modular and pattern-specific fashion. 
Type Of Material Database/Collection of data 
Year Produced 2020 
Provided To Others? Yes  
Title Research data supporting 'Pollen-feeding delays reproductive senescence and maintains toxicity of Heliconius butterflies' 
Description The dataset has information about Heliconius erato demophoon butterflies fed for 14d (young) and 45d (old) on three different diets: sugar only (N); sugar + supplement (C) ; sugar + pollen from flowers (F). These experiments were set up with 8 males and 8 females of similar size (~3 cm of forewing radius) per treatment (diet/age). At the end of the experiment, females were individually assay for fertility: number of laid eggs recorded and the total eggs per females collected for quantification of cyanogenic glucosides (CG) using target-metabolomic (HPLC-MS/MS). Recently ecloded butterflies (0d, unfed) was also collected as a baseline. All butterflies were also weighted and collected for target metabolomics at the end of the experiment. 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
Description ATAC-seq Heliconius consortium 
Organisation University of Puerto Rico
Country Puerto Rico 
Sector Academic/University 
PI Contribution The postdoc on the project worked in Panama during the project, collecting tissue and training local researchers in ATAC-seq methods
Collaborator Contribution The partner provided logistical support for butterfly rearing and conducting experiments
Impact ATAC-seq data set is still being analysed but we anticipate multiple publications resulting from this work.
Start Year 2018
Description Talks to schoolchildren 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Prof Jiggins gave three talks at St Johns College to visiting schoolchildren from London and Sheffield regarding evolution and genetics
Year(s) Of Engagement Activity 2019