Genetic mechanisms of amphibian colour pattern and toxicity in the natural environment

Overview

Colouration plays a key role in the ecology and life history of most species across all branches of the animal tree of life. Colouration and patterning are phenotypes that are strongly under selection for communication, thermal regulation, and/or crypsis. Amphibians present some of the most striking colour patterns found in animals, but the molecular mechanisms underlying this variation have remained among the most poorly understood of any vertebrate. This studentship research project will be the first to pinpoint and validate the molecular mechanisms of colour and toxicity in salamanders.

Fire salamanders endogenously produce poisonous secretions from specialised glands that they use in predator defence and are one of the only amphibians that can shoot their poison. Additionally, they are famously variable in colour and pattern across lineages and some populations are particularly variable (Fig. 1). Because of this combination of toxicity and striking colourations, fire salamanders are often considered a textbook example of aposematic colouration. It has also been argued that some colour variants are local adaptations to different environments, for example for thermoregulation. However despite a considerable body of research on salamanders over hundreds of years, the relationships between colour, the environment, and toxicity has not been established.

New ‘omics tools are opening a range of exciting possibilities in seeking to understand the adaptive functions of colour variation. For example, our recent work on fire salamanders studied an unusual geographic location where multiple colour morphs are found in sympatry, making it extremely powerful for genetic and ecological analysis. We identified molecular signals suggesting there is selection driving rare colourations. We also found significant associations between colour and genomic variation, indicating that colour is a genetically based trait and generating a suite of candidate genes for future, more detailed, investigation (Fig. 2). However, the evolutionary and ecological factors that drive these local variations in colouration have not been determined.

Fire salamanders in Europe are of major conservation concern. If the environment plays an important role on the evolution of such colourations, i.e. for thermoregulation, it is of outmost interest to understand the molecular basis of such mechanisms and their potential response to global warming that is predicted to affect salamander populations in the next decades.

This project will break new ground in our understanding of adaptation, chemical defence, and the genetic basis of colouration. Using Salamandra as a biological model, the research will ask:
1. Is colour variation associated with toxicity of skin secretions?
2. What are the environmental factors selecting for colour variation?
3. What is the genetic basis of colour and pattern variation?
4. Can colour variants be recapitulated using genome-editing?

To address these questions requires an integrative and multidisciplinary approach spanning genomic and evolutionary analyses, chemical analyses of venom, geographic information and landscape genomics, and colour variation in natural environmental context. This project will advance beyond correlational inferences to definitively test the genomic basis of skin colour-associated loci by cutting edge in vitro and genome-editing techniques.

With i) its cutting edge application of ‘omics and genome-editing on a fascinating natural model species of major conservation concern, ii) its addressing of an important and long-unanswered fundamental question in evolutionary biology and ecology, and iii) its expert supervisory team, this project has a strong likelihood of excellent high profile and high impact publications.

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Image Captions

used with permission, Elmer

Methodology

Fieldwork will be conducted in Europe (Iberian peninsula and the Alps) with PI Elmer and co-I Vieites. Some genetic material is already in hand from previous research and current populations of captive-reared salamanders, which will provide an efficient starting point. There will be extensive opportunity for the student to develop and refine field, phenotyping, and molecular approaches.

Genetic analyses will be conducted using advanced genome-wide next-generation sequencing approaches. Salamanders have exceptionally large genomes but modifications exist that can cost-effectively sequence the coding portion of the genome, which is then no longer particularly large. This is made possibly by the existing resources and expertise established in the PI’s group and the affiliated research community.

Analytical methodologies will aim to identify significant association of loci with colour phenotype, using Bayesian and machine learning approaches. Environmental information will be combined with field data on habitat and altitude. Population level analyses will query genomic-phenotypic-environmental co-variations to infer ecological and evolutionary associations. The student will work closely with the co-Is in St Andrews for advanced genomic-environmental data analysis, including an extended research visit during data analysis in year 2.

Individual-level metabolomic profiling will be conducted on salamander skin toxins to infer the steroid chemical composition of the secretions using gas chromatography-mass spectrometry. Comparisons between colour and biochemical profile of the secretions will be conducted across multiple population replicates. The student will work with co-I Burgess to characterise and analyse metabolomics profiles.

Genome editing will be done on species-specific skin cell culture. Research affiliated with the PI is currently developing in vitro culture of relevant tissues. Drawing on a compelling list of candidate genes inferred from this research, targeted knock-outs will be generated by CRISPR/Cas approaches. The student will lead this exciting new research direction supported by the supervisory team and the momentum currently underway in the Research Institute and the PI and co-I’s lab in St Andrews.

Project Timeline

Year 1

Molecular lab work and sequencing; data analysis; field collections; tissue culture establishment.

Year 2

Analysis of genomic-phenotypic-environmental relationships; metabolomics data collection; genome-editing on tissue culture

Year 3

Analysis of metabolomics and genomics; genome-editing validation

Year 3.5

Data analysis; dissemination of results by high impact publications and presentation at conferences.

Training
& Skills

The student will train with internationally esteemed researchers to acquire a breadth of skills necessary to pursue a career in quantitative biology: amphibian fieldwork, colour analysis in the visible and non-visible spectrum, skills in molecular biology for high throughput sequencing, tissue culture and learn the cutting edge genomic approaches for understanding relationships between phenotypic and genotypic variation (Fig. 3). In addition, generic training in transferable skills will be a core component (e.g. experimental design, statistics, oral and written communication) and are well supported by the outstanding graduate programme at Glasgow.

The project will be based in Glasgow, where the student will join a successful, active, and vibrant research community at an institution of high national and international standing. The data analysis and communication skills gained in this project will have key relevance for employability in conservation, academia, biotech, or government research.

References & further reading

Karageorgi M, et al. (2019) Genome editing retraces the evolution of toxin resistance in the monarch butterfly. Nature. 574, 409-412.

Lueddecke T, et al. (2018) A salamander’s toxic arsenal: review of skin poison diversity and function in true salamanders, genus Salamandra. The Science of Nature. https://doi.org/10.1007/s00114-018-1579-4

Crawford NG et al. (2017) Loci associated with skin pigmentation identified in African populations. Science, 358, eaan8433.

Rodríguez A et al. (2017) Inferring the shallow phylogeny of true salamanders (Salamandra) by multiple phylogenomic approaches. Molecular Phylogenetics and Evolution, 115, 16.

Beukema W, Speybroeck J, Velo-Anton G (2016) Quick guide: Salamandra. Current Biology, 26, R689.

Burgess, K, et al. (2011) Semi-targeted analysis of metabolites using capillary-flow ion chromatography coupled to high-resolution mass spectrometry. Rapid Communications in Mass Spectrometry 25: 3447.

Foll M, Gaggiotti O (2008) A genome-scan method to identify selected loci appropriate for both dominant and codominant markers: A Bayesian perspective. Genetics, 180, 977.

Further Information

Applications: to apply for this PhD please use the url: https://www.gla.ac.uk/study/applyonline/?CAREER=PGR&PLAN_CODES=CF18-7316

For questions contact Dr. Kathryn Elmer, Kathryn.Elmer@glasgow.ac.uk or +44 141 330 6617

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