Genetics and Evolutionary Ecology of White Clover (Trifolium repens)

Readings - Clover background - Exercises -  Questions / Homework - For Instructors (methods)


Hayden, K.J. and Parker, I.M. 2002.  Plasticity in cyanogenesis of Trifolium repens : inducibility, fitness costs and variable expression. Evolutionary Ecology Research. 4: 155-168.

Clover Background Information

(From Antonovics, 1973. Practical Genetics.  P.M. Sheppard (ed). Blackwell Scientific Publications: London. pp 38 -)

(i) White clover (Trifolium repens)
White clover is of special interest, since its populations are polymorphic for several sets of major gene characters. Clover can be easily grown from cuttings (a few internodes long) pushed into ordinary garden soil or potting compost. It thrives best in cool bright conditions, and the leaf mark characters in particular are seen most clearly outdoors in spring, or in a cool greenhouse with artificial light. These conditions also reduce the chances of infection from aphids or red spider - an important consideration since greenhouse sprays and insecticides can easily damage clover leaves. Clover grows quickly and has to be replanted at least once a year, or cut back to prevent stolons trespassing into adjacent pots. Unwanted seed heads should also be removed before they are ripe, to prevent foreign seed germinating and "contaminating" the existing plates.

Clover seed germinates slowly, and it is best scarified (weakening of seed coat) by immersing dry seed in conc. H2SO4 for 15 min, draining off the acid, and washing with plenty of water. Alternatively the seed can be rubbed with sandpaper. The seed is then sown directly or germinated on a moist filter pad and then sown as a small seedling.

The seedlings will flower if they are given summer (long) day length, but plants which have flowered once or adult plants collected in the field need short days and/or cold treatment (outdoors in winter) for about two months, followed by long days for flowering to occur. In natural conditions the plants normally flower between  May and July. Clover is to all intents and purposes self-sterile so emasculation is unnecessary. Pollen is best transferred using the pointed tip of a cardboard triangle, which has been folded down the middle (see fig 2.2). The keel is pulled away from the head with a pair of fine forceps and the stigma is extruded for pollination, The plants or flowerheads can be covered in muslin to prevent stray pollination, or an insect-free greenhouse should be used.  About four seeds per floret are produced after 4-6 weeks. Pollination is made easier if the number of florets per head is reduced to about 20 which are at an equivalent stage with their wing petals about to open.


The ability to release cyanide when the plant is damaged is determined by two unlinked genes, and populations of clover are normally polymorphic for both genes. One gene, Ac, determines the ability to produce the glucosides, linamarin and lotaustralin, which will liberate HCN when acted upon by an enzyme. The presence of this enzyme, linamarase, is determined by another gene, Li. Both genes are dominant (to inability to produce glucoside and enzyme), and both have to be present for full cyanide production.
The normal test for cyanide-production is as follows. Crush and macerate two or three leaves in a few drops of water at the bottom of a small 50 mm x 15 mm glass tube (using a glass rod). Add two drops of toluene and insert a short slip of sodium picrate paper by jamming it in between the cork and the side of the tube. The paper should be clear of the liquid at the bottom. The tubes are then incubated for 2 hours at 40o C, and if cyanide is released it will turn the sodium picrate papers from yellow to a reddish brown. This will only be positive for the Ac Li plants.

However, Ac Li plants can be distinguished from Ac li because spontaneous  (non-enzymatic) hydrolysis of the glucosides  also takes place but more slowly. If the tubes are left for at least another 24 hours, Ac li plants will also turn the sodium picrate paper brown.

The ac Li plants can only be detected by adding glucosides to see if the enzyme is present to break them down. Both Ac li / li and Ac Li / - plants can be used, because if the leaves are autoclaved  (110  oC for 25 min) then the enzyme but not the glucoside is inactivated.  About 100 leaves should be macerated in 100 mL of water, the extract filtered, and two drops (instead of two drops of water) used to macerate the cyanogenic leaves to test for Li. The preparation should be made just prior to the tests, as it keeps its activity for a few days only.

Once recognized by the appropriate tests, the genotypes can be propagated vegetatively. Crosses between completely acyanogenic and cyanogenic types will serve to identify the genotypes of the cyanogenic forms, and can serve as a backcross. Once these genotypes have been recognized, F2 ratios can be generated by crossing genotypes heterozygous for either one (to give a 3:1 ratio) or both genes. If no attempt is made to distinguish the non-cyangogenic types, a 9:7 ratio is obtained, but if Ac li, ac Li and ac li plants are distinguished, a 9:3:3:1 ratio will be obtained. This is a good example of evidence of complementary gene action since both genes are necessary for the biochemical sequence to take place, and therefore for the character to be expressed.

The value of these various investigations in clover is that they provide a link between elementary genetics, population genetics and natural selection. The cyanogenesis character is also a clear demonstration of the way in which genes act in metabolism.

Evolution of herbivory defense.

Introduction: (For references see:  Hayden, K.J. and Parker, I.M. 2002.  Plasticity in cyanogenesis of Trifolium repens : inducibility, fitness costs and variable expression. Evolutionary Ecology Research. 4: 155-168.)

Hydrogen cyanide (HCN) production is an anti-herbivore defense present in many species of plants. In white clover (Trifolium repens) cyanogenesis is maintained as a polymorphism. It has been assumed that cyanogenesis benefits the plants in the face of herbivory, since several small herbivores (snails and voles) have been shown to prefer acyanogenic to cyanogenic plants. Implicit in the existence of a genetic polymorphism, however, is that there is a cost to cyanogenesis (otherwise all individuals would have the trait).

Studies have suggested that there is a positive correlation between altitude and proportion of acyanogenetic plants. Whether this correlation is a consequence of  lower herbivory at higher altitudes or lower temperatures at higher altitudes has previously been unclear. It has been suggested that production of hydrogen cyanide decreases ability of plants to tolerate low temperatures and drought.  It has also been unclear whether there is a plastic response in individual plants to upregulate cyanide production in the face of herbivory - though it now appears that individual plants do not change cyanide production after leaf damage of real and simulated herbivores.

Possible lab and field experiments with clover and herbivory defense .

1) Induced defense: does simulated (cutting) or real herbivory (slug/snail) or environmental extremes (freezing or heating) trigger cyanide production in clover. (Hayden and Parker suggest no)

2) Is there a correlation between elevation (which may mean changes in temperature or relative levels of herbivory) and proportion of clover in a population that is cyanogenic? This exercise could be conducted on a field trip by collecting clover at various elevations and then examining the HCN content.

3) What is the cost of cyanogenesis? (some suggestions include : acyanogenic plants have higher drought and cold tolerance) - trade-offs with a beneficial trait - (why are there acyanogenic plants to begin with?). A laboratory experiment could be conducted to test cyanogenic and acyanogeinc morphs for temperature extreme tolerance or drought tolerance.

4) Choice experiments: give herbivores a choice between cyanogenic and acyanogenic plants (snails are probably easiest to use - though voles have been used in experiments before). Requires testing clones of clover for cyanogenesis and then cultivating clones.

Discussion questions / Homework Assignment:

  1. Does the existence of a genetic polymorphism imply a cost to the trait? Explain. 
  2. What are the evolutionary implications for a population that has inducible defenses (individuals can up or down regulate a defense depending on need) versus a genetic polymorphism in which some individuals have the trait while others do not?
  3. Can plasticity itself be a selected trait? How would this work?

Listed below is a detailed protocol for testing for cyanide content from the  Colorado State Cooperative Extension.

Testing clover samples for cyanide content 

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Vol. 1 No. 3 February 16, 1981


I. Cyanide and Nitrate Cyanide

Cyanide (Prussic Acid) poisoning may occur in domestic animals when they ingest plants which contain large quantities of cyanogenetic glycosides. Cyanogenetic glycosides are sugar complexes in which the cyanide molecule is chemically tied up so that it cannot exert a toxic effect. There are two basic ways in which the cyanide in the cyanogenetic glycosides can be released and cause toxicity.

  1. Damage to the plant cells may release plant enzymes which cause the release of cyanide from the glycoside complex. Weather conditions such as drought and frost can initiate this process.
  2. The action of enzymes in the digestive system of an animal species. Ruminants are particularly susceptible to this.

Plants which have the potential to cause cyanide poisoning in domestic animals are as follows: Sorghum species, white clover, arrow grass, corn, flax, lima beans, leaves and pits of Prunus species (cherries, apricots, peaches) and Apple seeds. The clinical signs of cyanide intoxication in domestic animals include excitement, muscle tremor, rapid labored breathing, collapse, convulsions, and death. A simple reliable field test is available which will detect the presence of toxic quantities of cyanogenetic glycosides in plants.

Rapid Field Test for the Detection of Cyanide

  1. To a large test tube or a small flask, add 1 to 5 g. of finely chopped fresh plant material or rumen contents. If the material is dry or you want to check seeds, macerate it with a little water.
  2. Add 4 to 12 drops of chloroform.
  3. Prepare the picrate test strip by putting a drop of the picrate solution on a strip of filter paper. Close the container and hang the picrate strip so that it does not touch the sides or the material at the bottom of the tube.
  4. Warm the container to 30 to 37C. Let reaction continue about three hours. The test is positive if the strip turns various shades of red. The appearance of a dark brick red color is significant. Milder color changes that occur are of little concern.
  5. A negative test suggests the absence of a cyanogenetic glycoside, or that a hydrolyzing enzyme was not closely associated with it.

Picric Acid Test Solution

  1. Sodium Bicarbonate, 5 gm., and picric acid, 0.5 gm., QS 100 ml. with water. Keep cool in glass-stoppered brown bottle.
  2. Try the test using 5-10 apple seeds as a positive control. This will give an idea of the type of color change that is significant.

The test procedures listed in this newsletter are modifications of a method published in Clinical and Diagnostic Veterinary Toxicology, Second edition, edited by G.A. vanGelder, Kendall/Hunt Publishing Company, Iowa, 1976.

Arthur L. Craigmill, Ph.D.
Extension Toxicologist
Environmental Toxicology and Veterinary Extension
University of California
Davis, CA 95616
(530) 752-1142

Test for Prussic Acid
This is a qualitative test to evaluate forages (hay, pasture, silage) for prussic acid poisoning potential in ruminants.

© Colorado State University Cooperative Extension. 1995-2003.
Contact Cooperative Extension Web Manager.
Home Page:

Issued in furtherance of Cooperative Extension work, Acts of May 8 and June 30, 1914, in cooperation with the U.S. Department of Agriculture, Milan A. Rewerts, Director of Cooperative Extension, Colorado State University, Fort Collins, Colorado. Cooperative Extension programs are available to all without discrimination. No endorsement of products mentioned is intended nor is criticism implied of products not mentioned.