A direct experimental test of Ohno’s hypothesis

• Background to the preprint.

Gene duplications are a common evolutionary phenomenon. However, comprehending the underlying mechanisms, identifying the resulting patterns, and determining the circumstances that lead to these patterns remains a challenge. Multiple hypotheses have been proposed, yet these theories have seldom been linked to experimental models. According to Innan and colleagues, gene duplication can be classified into three main categories: category I includes neofunctionalization and subfunctionalization of the duplicated genes, where duplication is neutral with no selection acting before the duplicate reaches fixation; in the case of category II, the gene duplication is advantageous (adaptive); category III gene duplication is advantageous but occurs in a polymorphic gene that becomes fixed in the duplicated copy.

This preprint focuses on Ohno’s hypothesis (category I), which proposes that a duplicated gene is relaxed from purifying selection and thus can result in pseudogenization or gain a new function by accumulating mutations (Fig1). Some evolutionary predictions arising from this model involve equal divergence before fixation and combined copy gene functions contributing to fitness, similar to unduplicated orthologs in closely related species.

• Key findings of the preprint.

-Under selection, the redundancy provided by gene duplication appeared to enhance gene resilience to mutations observed during “early” time points (generations 1-3). This was evaluated based on robustness, measured by the number of cells that maintained fluorescence after mutagenesis over multiple generations.

-Gene duplication did not lead to enhanced early functional differentiation. To test Ohno’s prediction that gene duplication facilitates phenotypic evolution, the authors searched in the green-selected experiment whether green fluorescence evolved faster in double-copy populations. In contrast to the prediction, the authors observed no difference in fluorescence but noticed greater variance in double-copy populations.

-Gene duplication leads to a rapid loss of function before diverged gene function. The experiment showed that after one generation of double-copy populations around ~40% of cells possessed an inactive copy.

-In agreement with other preprint results, duplicated gene copies diversified faster on average than control single active and inactive copies. Although most mutations are under purifying selection, the experiment also showed that some common mutations arise frequently that may confer structural protein stability.

-Early loss of function in either gene copy might explain enhanced purifying selection on the active one, reducing the relative differences with single copy genes. The inactivation of one copy leads to higher constrained in the remaining copy; thus, making single copy and double copy populations (after inactivation of one copy) behave similarly after the 3^rd generation in this experiment.

• What I like about the preprint/why I think this new work is important.

What I like about this preprint is that it tests a fundamental theoretical biological framework possibly driving common evolutionary patterns: gene duplications. It is hard to find experimental studies testing evolutionary hypotheses in the literature. Although the model presented in this work lacks biological realism, it is capable of testing Ohno’s (evolutionary) hypothesis. Also, observational approaches, including comparative analysis, that have been used in the past agree with the results presented in this preprint. These usually lack experimental validation but in this preprint the authors have set up a clever experimental evolution design demonstrating that their approach can reconcile theory and data, making evolutionary hypotheses and their predictions testable.

• Questions for the authors

If selection pressure was constant across generations or if the direction was variable across generations what would be your prediction for this gene duplication system?

Could you please elaborate on why you think only green specialization was observed? why not a symmetrical behavior when applying similar selecting pressures to both phenotypes?

In the future, would it be possible to predict the time window for the gene to lose or gain a new function? Could you adapt your experimental system to make inferences about it?

Using your system, could you speculate on the time window between gene duplication innovation and loss of function? Do you think this is mostly driven by gene-specific characteristics or population-driven effects?

Could your system be expanded to predict whether “protein” promiscuity precedes a specific duplication hypothesis?

• References

Innan, H., & Kondrashov, F. (2010). The evolution of gene duplications: Classifying and distinguishing between models. Nature Reviews Genetics, 11(2), 97-108. https://doi.org/10.1038/nrg2689

 

doi: https://doi.org/10.1242/prelights.36041

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