Reconstruction of Pollinator-Mediated Speciation by Identification and Substitution of Causative Mutations

Project Information

RESPEC

Grant agreement ID: 741354

DOI

10.3030/741354

Project closed

EC signature date 22 May 2017

Start date 1 September 2017

End date 31 December 2022

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 2 499 380,00

EU contribution

€ 2 499 380,00

Coordinated by

UNIVERSITAET BERN
Switzerland

Periodic Reporting for period 4 - RESPEC (Reconstruction of Pollinator-Mediated Speciation by Identification and Substitution of Causative Mutations)

Reporting period: 2022-03-01 to 2022-12-31

Summary of the context and overall objectives of the project

A central idea of evolution is that it is gradual. Evolution, not revolution. In genetic terms that means that evolution proceeds by an infinite number of mutations of infinitely small effect. Of course, we now know that the number of genes in a typical eukaryote is not infinitely large, but rather in the order of 25,000. Still, many adaptations probably do require multiple mutations, each of small effect. However, especially when selection is strong and reproductive isolation incomplete, evolutionary change through few major mutations each of large phenotypic effect might be possible. A promising system to test this hypothesis is pollinator-mediated speciation. If, for whatever reason, an established pollinator does not show up when its host is about to start flowering, the plant is under strong selective pressure to rapidly adapt to an alternative pollinator.

In RESPEC we used wild Petunia species that are adapted to different pollinators: bees, nocturnal hawkmoths and hummingbirds. We asked which genes needed to mutate to make a flower attractive to a hummingbird or a bee instead of a hawkmoth. We started with the simplest system, moth-pollinated to bee-pollinated, because these plants have very similar morphology and differ in so-called attraction traits, the color, UV absorption and scent of the flower. We identified four “major genes” that are responsible for most of the differences between the two species in visible color (1 gene), UV absorption (1 gene) and scent emission (2 genes). We started with the moth-pollinated plant and changed these four genes in such a way that they are functionally identical to their counterparts in the bee-pollinated plant. Then we tested whether moths and bees can tell the difference between this artificial mimic and the wild model plant. Our results indicate that they cannot.

People often ask why we are doing this. Is it because of the worldwide decline in bees? Could we change fruit trees to make them attractive to different pollinators? To be honest, I have my doubts about whether that is such a great idea. The motivation was really the challenge to work on something that had never been done and that was long thought to be impossible.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

We started with floral UV-absorption. Hawkmoth-pollinated flowers absorb UV light and the moths have photoreceptors for UV. As a consequence, even at very low light intensity, the flowers stand out against the rest of the vegetation. For bees, UV is not essential and bee-pollinated Petunias don’t produce UV-absorbing compounds. We used CRISPR/CAS9 to knock out the single gene that is important for UV absorbance. The interesting surprise was that the flowers not only became UV-reflective but also started producing visible pigments, the flowers turned pink, a color preferred by bees. Behavioral assays demonstrated that hawkmoths strongly prefer the white, UV absorbing parent, but that bees are drawn to the UV-reflective flowers. Mutating the genes that cause the differences in visible color and scent is in progress.
The evolutionary transition from moth to hummingbird pollination is more complex. We zoomed in on the genes that cause the differences in flower morphology using a variety of complementary methods. We made substantial advances with the origin of the typical red “hummingbird color”. A regulatory gene called DeepPurple (DPL) needed to be modestly upregulated for making the red pigments. Strong overexpression of DPL in the hummingbird plant resulted in a purple color. The big surprise was that such DPL-overexpression plants lost their specialized “hummingbird morphology” and reverted almost completely to the moth morphology. We hope that this phenomenon will shed light on the evolution of hummingbird flower morphology.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The UV-CRISPR mutant shows that a single mutation has the expected major effect on floral UV-absorption, but unexpectedly, also on visible color. Behavioral experiments in which wild parent and CRISPR mutant are exposed to hawkmoths or bees demonstrate that this single genetic change has a major effect on pollinator preference. We obtained mutations in the other three genes and combined them in a single background. Our results show that bees cannot tell the difference between this artificial mimic and the wild model plant.

Knockout of MYB-FL affects both UV absorption and visible color