Cyanogenesis in White Clover

Genetic and physiological mechanisms of local climatic adaptation in a widespread perennial plant species.

NSF IOS-1557770


Adaptation to local environments plays a key role in the ability of plant species to survive in various ecological settings and persist through climate change, yet the genetic and physiological mechanisms that underlie this process are largely unknown. This research examines the basis of local adaptation in a widespread perennial that is considered a textbook example of adaptive climatic differentiation, where fitness consequences of natural biochemical variation can be directly assessed in both the lab and field.

White clover (Trifolium repens L.) has evolved climate-associated differentiation in cyanogenesis (HCN release with tissue damage) in populations worldwide. Our previous work has determined the molecular basis of cyanogenesis variation and documented adaptive differentiation across climatic gradients in aridity and winter temperature. Preliminary data suggest that gene copy number variation (CNV) at cyanogenesis genes contributes to this local adaptation. While there is strong evidence that cyanogenesis serves as an herbivore deterrent, there is growing evidence from crop species that cyanogenic precursors may also function in abiotic stress tolerance. Putative functions, which include nutrient storage and transport, osmoprotection, and oxidative stress mitigation, remain uncharacterized, and their potential adaptive significance in nature is untested. White clover is genetically tractable and easily manipulated through field experiments in the environments where adaptive differentiation has evolved, making it highly suited for testing hypotheses on the genetics and physiology of cyanogenesis and climatic adaptation.

Through work that integrates QTL mapping of fitness traits, candidate gene analyses of two well-characterized biochemical polymorphisms, and physiological assessments of their fitness impacts, the following questions are addressed: 1) What is the genetic architecture of local climatic adaptation? Specifically, to what extent does it arise through fitness tradeoffs for alternate alleles of individual genes (antagonistic pleiotropy), or through combinations of loci where individual genes provide a fitness benefit in one environment and are selectively neutral in others (conditional neutrality)? 2) What are the physiological mechanisms by which cyanogenic glycosides function in abiotic stress adaptation? 3) What is the role of gene copy number variation (CNVs) in local adaptation?

Intellectual merit:

There is increasing evidence that plant secondary compounds may play important roles in primary metabolism. This work will rigorously test the function of cyanogenic glycosides, which are found in >3,000 plant species including many crops, in abiotic stress adaptation. At the genetic level, local adaptation is often assumed to occur through antagonistic pleiotropy, and this assumption forms the basis of popular genome scans for detecting genes under selection. Yet very few studies have tested this assumption in nature. This research will determine the contribution of antagonistic pleiotropy to climatic adaptation, and it will assess of the importance of well-characterized candidate gene polymorphisms in relation to other genetic factors. CNVs are abundant in the genomes of many taxa, and there is growing evidence that they may play an important role in natural adaptive variation; remarkably, their fitness impact in natural populations has never been directly assessed, as will be done in this research.

Broader impacts:

The ability of plant populations to adapt to climatic variation will be critical for continued worldwide crop productivity and for persistence of wild species through climate change; this research provides basic insights into the mechanisms by which climatic adaptation occurs. The project will engage high school students and teachers nationwide in hands-on, inquiry-based learning through an ongoing and highly successful outreach program based on clover cyanogenesis. Students sample and analyze their local clover populations and contribute data to an online database, where they can compare observations with classes from other climates. Undergraduates will be recruited for internships providing training in lab and field components of plant biology and evolutionary genetics; students from under-represented backgrounds will be targeted for recruitment through the WU uSTAR and UF Agronomy programs. The project will provide training in plant physiology and evolutionary genomics for two graduate students and two postdocs, and it will foster new academic partnerships between four U.S. research institutions.



Clover cyanogenesis: integrating ecological and molecular genetics in the study of adaptation.

NSF CAREER award, DEB-0845497

Molecular evolution of cyanogenesis in white clover (Trifolium repens)

White clover (Trifolium repens) is naturally polymorphic for cyanogenesis (HCN production with tissue damage). Cyanogenesis protects plants from small herbivores, but frequencies of cyanogenic plants decrease in colder climates, possibly because cyanogenesis is detrimental to plants in areas of frequent frosts. The cyanogenesis polymorphism is controlled by two genes: Ac controls the presence/absence of cyanogenic glucosides, and Li controls the presence/absence of the enzyme required for their hydrolysis (linamarase). We are currently examining the molecular evolution of the cyanogenesis loci to understand the genetic basis and evolutionary dynamics of this adaptive variation.

CAREER award summary:

Objectives - Understanding the genetic basis of adaptation is a central goal of evolutionary biology. An exciting advance of the last decade has been the development of studies characterizing the molecular basis of adaptive polymorphism. Most such studies have focused on model organisms, where little is known about the connection between phenotypes and fitness in natural populations - an essential connection for the study of adaptation. This CAREER project capitalizes on an ecologically well-characterized system to study the molecular evolution of an adaptive polymorphism within its native ecological context. White clover (Trifolium repens) is polymorphic for cyanogenesis (cyanide release with tissue damage). This polymorphism has been the subject of extensive ecological study for over sixty years, making it one of the best documented cases of an adaptive polymorphism in plants. I have recently characterized the molecular genetic basis of the cyanogenesis polymorphism, and I have also documented the polymorphism's occurrence in several related species. This makes it possible to now integrate molecular and ecological genetics in this system to examine the origin and evolution of adaptive polymorphism. Three specific questions are addressed:

  1. Molecular origin of intraspecific adaptive polymorphism. Do adaptive phenotypic polymorphisms correspond to longstanding balanced polymorphisms at the molecular level? Or have the alleles underlying adaptive phenotypes evolved multiple times independently?
  2. Evolution of transspecific polymorphism. Do polymorphisms that transcend species boundaries reflect balancing selection to maintain ancient allele lineages? Or have polymorphisms evolved independently among species?
  3. Selective maintenance of polymorphism in contemporary populations. What are the relative roles of coarse-grained environmental heterogeneity and within-population balancing selection in shaping the distribution of this adaptive polymorphism? Specifically, what role does epistatic selection play in maintaining the polymorphism within populations?

Broader Impacts - Many of the features that make clover cyanogenesis attractive for research also make it highly suited for use as a teaching tool in the Biology classroom: the polymorphism follows simple Mendelian genetics; the trait is easily visualized through a simple colorimetric assay; clover plants are easily obtained from school lawns and parks nationwide; and the key ecological factors maintaining the polymorphism are well documented and understandable at the high school level (herbivore defense, energetic tradeoffs). This CAREER project uses a multifaceted approach for developing the clover cyanogenesis system an educational resource:

  1. High school teacher summer lab internships. Lab internships not only provide opportunities for science teachers to experience research, but they also allow teachers to put their own expertise to work in optimizing lab protocols for use in the classroom. Four summer lab internships will be conducted, involving both Introductory and Advanced Biology teachers.
  2. Integration of clover cyanogenesis into the LSGC Summer Teacher Institute. The Life Sciences for a Global Community summer institute is an NSF-funded program at Washington University that trains Biology teachers from around the country towards a MS-Biology degree. I will integrate a clover cyanogenesis lab into the LSGC curriculum, providing a national cadre of 90 teachers with training on the use of this system as a teaching tool.
  3. Development of a "clover cyanogenesis kit" and supporting web-based resources. A lab kit for use in the classroom and related online teaching resources will be developed and disseminated both nationally through the LSGC and to teachers in the St. Louis community. The website will feature a database of cyanogenesis frequency data, collected by students nationwide and entered online. This method of data collection will allow students to be actively engaged in scientific research, and the data will provide a wealth of information for research questions on the geographical distribution and selective maintenance of the cyanogenesis polymorphism.

If you are a high school science teacher interested in using clover cyanogenesis as a teaching tool in your classroom, or if you are a high school or university student interested in trying this out as an independent project, please see "The Clover Project" website for more information and materials.