IAP2-22-426

What are the genetic targets of sexual selection underlying rapid phenotypic divergence among species?

Background
Sexual selection can drive the rapid divergence of male secondary sexual traits, including bird plumage and the external genital structures of insects (Eberhard 2010). Identifying the genes and mutations that cause these differences is key to understanding the role of sexual selection as a driver of phenotypic diversity among species. However, the genetic basis of these differences is poorly understood (Hagen et al., 2019). The external male genital structures of flies are derived from legs and play important roles in copulation (Casares et al., 1997). For example the posterior lobes of the male genital arch intercalate between the female tergites and are important for species-specific recognition and copulation (Frazee and Masly, 2015). The posterior lobes of males of species of the Drosophila simulans clade (D. simulans, D. sechellia and D. mauritiana) have diverged dramatically in size and shape in less than 240,000 years, and are the only way to reliably distinguish these species morphologically (Garrigan et al., 2012; Kopp and True 2002). This difference is polygenic in basis but none of the causative genes have yet been identified (Zeng et al., 2000; Hagen et al., 2021). This project aims to identify and test genes underlying posterior lobe divergence through the following experimental objectives.
O1. Identify differentially expressed candidate genes underlying posterior lobe divergence between D. simulans, D. sechellia and D. mauritiana.
O2. Explore sequence variation of candidates within and among species.
O3. Test the function of candidates during posterior lobe development.
O4. Characterise the effects of variation in candidate genes on mating behaviour and reproductive isolation.

Methodology

O1 will compare RNA-seq data among the developing genitalia and legs of D. simulans, D. sechellia and D. mauritiana to identify genes that are differentially expressed in the genitalia but not legs that thus represent candidate genes contributing to differences in posterior lobe morphology among these three species.
O2 will use population genetics data to examine the levels of segregating and fixed sequence variants of candidate genes within and between the three species to help infer their contribution to posterior lobe divergence.
O3 will apply CRISPR/Cas9 genome editing to generate reciprocal hemizygotes to test the function of candidate genes and verify their contribution to posterior lobe divergence.
O4 will use behavioural tests to compare the effect of candidate gene reciprocal hemizygotes on different aspects of copulation and fitness. These experiments will be carried out at Oxford Brookes University supervised by Dr Daniela Nunes.

Project Timeline

Year 1

Months 1-12: Bioinformatic analysis of RNA-seq data to identify candidate genes (O1) and commence analysis of their sequence variation (O2).

Year 2

Months 1-8: Completion of O1 and O2 to identify candidate genes and shortlist for functional (O3) and behavioural analysis (O4).
Months 6-12: Design and cloning of constructs for CRISPR/Cas9 to generate reciprocal hemizygotes for up to 5 genes for O3 and O4.

Year 3

Months 1-10: Generation and phenotypic analysis of reciprocal hemizygotes of candidate genes (O3).
Months 4-12: Behavioural analysis of candidate gene reciprocal hemizygotes (O4).

Year 3.5

Months 1 and 2: Completion of behavioural tests (O4).
Months 2-6: Thesis writing.

Training
& Skills

The student will be trained by three supervisors with complementary experience and skills in different areas of wet lab, computational and theoretical biology. This will be enhanced by interactions with other PhD students and postdocs in their lab groups and other groups at all three organisations. The student will participate in and present at lab meetings and journal clubs, and through regularly individual meetings with all three supervisors on zoom. The student will attend a conference annually and appropriate workshops/summer schools to expand their training. Opportunities will also be provided for the student to supervise undergraduate projects. All three supervisors have established outreach programmes that the student will also contribute to. This training programme will furnish the student with skills and experience in a range of experimental approaches and theory in evolutionary biology, population genetics, bioinformatics, developmental biology, molecular biology and animal husbandry and behaviour. The student will also develop their transferable skills in data analysis and management, problem solving, oral and written presentation, project management and supervision.

References & further reading

Casares et al. (1997). The genital disc of Drosophila melanogaster I. Segmental and compartmental organisation. Dev. Genes Evol. 207: 216
Eberhard (2010). Evolution of genitalia: Theories, evidence, and new directions. Genetica 138: 5.
Frazee and Masly (2015). Multiple sexual selection pressures drive the rapid evolution of complex morphology in a male secondary genital structure. Ecology and Evolution 5:4437.
Garrigan et al. (2012). Genome sequencing reveals complex speciation in the Drosophila simulans clade. Genome Res. 22: 1499.
Hagen et al. (2019). tartan underlies the evolution of Drosophila male genital morphology. PNAS USA 116:19025.
Hagen et al. (2021). Unravelling the genetic basis for the rapid diversification of male genitalia between Drosophila species. Molecular Biology and Evolution, msaa232, https://doi.org/10.1093/molbev/msaa232.
Kopp and True (2002). Evolution of male sexual characters in the oriental Drosophila melanogaster species group. Evolution and Development 4: 278.
Zeng et al. (2000). Genetic architecture of a morphological shape difference between two Drosophila species. Genetics 154: 299.

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