In White Clover (Trifolium Repens L.)

Heredity 59 (1987) 265—271 TheGenetical Society of Great Britain Received 25November 1986

Variability of expression of cyanogenesis in white clover (Trifolium repens L.)

Irene Till CNRS-CEPE, 34033 Montpellier Cedex, France. The pattern of inheritance of the polymorphism of cyanogenesis in white clover, i.e., of two independent loci with a pair of alleles at each locus, is shown to be too simple. Several phenotypes can coexist at the same time in the same plant, without showing any detectable pattern in space or time. A study of the inheritance of this intra-individual heterogeneity showed that homogeneity is dominant over heterogeneity and that the heterogeneity of a plant increases with the number of its heterogeneous parents. The ecological and evolutionary significance of the phenotypic heterogeneity is discussed.

INTRODUCTION 1980; Dirzo and Harper, 1982). Thus there are nine possible genotypes and four phenotypes that can Aplant is said to be cyanogenic when it releases be readily identified by appropriate tests (Corkill, hydrogen cyanide (HCN) from damaged tissues. 1940), giving F2 ratios of 9:3 :3: 1 for plants having Cyanogenesis in the leaves of Trifolium repens L., both the glucosides and the enzyme (GE), only white clover, results from the hydrolysis of two the glucosides (G—), only the enzyme (-E) and cyano-/3-glucosides (linamarin: 2-hydroxy- neither (--) andof 9:7 for cyanogenic (GE) to isobutyronitrile-f3-D-glucopyranoside; lotaus- acyanogenic (G-, -.E and .--)plants. tralin:(R)-(hydroxy-2-methylbutyronitrile-/3-D- A similar pattern of inheritance has been glucopyranoside)) by a specific f3-glucosidase observed with Lotus cornicultus L. although this (linamarase). The two cyanoglucosides are synthe- plant is a tetraploid showing tetrasomic inheritance sized from valine and isoleucine respectively (e.g., at these two loci (Dawson, 1941; Bansal 1971; Cutler and Conn, 1982) by the same biosynthetic Ramnani and Jones, 1984). pathway (Collinge and Hughes, 1984). In intact Quantitative variation in HCN production has leaves the substrates and enzymes are kept separ- frequently been recorded (Doak, 1933; Williams, ate. It is known that the linamarase is located in 1939; Daday 1954a; Jones, 1966). In T. repens the cell walls of epidermal cells in the leaves of T. there is a clear allele dosage effect, the homozygous repens (Boersma et a!., 1983; Kakes, 1985). It is AcAc individuals producing, on average, more only surmise from other work that the cyanogluco- than twice the mass of the cyanoglucosides that sides may be stored in the vacuoles of these cells can be found in heterozygous Ac ac plants (Hughes (Kojima et a!., 1979; Cutler and Conn, 1982). and Stirling, 1982). From formal breeding work Corkill (1942) pro- All four phenotypes can be found in almost all posed that the inheritance of cyanogenesis involves populations of T repens (e.g., Daday, 1954a, b) two independent loci each with two alleles. For and therefore the species is polymorphic at both each locus the dominant allele determines the pres- loci. In attempts to explain this genetic polymorph- ence of either the glucosides (Ac) or the enzyme ism, two selective forces have been invoked: (Li). This interpretation has been widely accepted (a) Herbivory: It has been shown with T. repens (Daday, 1954a, b, 1958; Chakravarty, 1963; De (Dirzo and Harper, 1982), with L. corniculatus Arraujo, 1976; Cahn and Harper, 1976; Burdon, (Jones, 1962, 1966, 1972 and with both species (Crawford-Sidebotham, 1972) that Present address: GPDP-CNRS, 91190 Gif Sur Yvette, France. slugs and/or snails prefer acyanogenic to 266 I. TILL cyanogenic plants when given the choice. mechanisms having differential effect on these Furthermore, the molluscs do not differentiate three phenotypes. between the three types of acyanogenic (G-, During studies on the distribution of —E or --)plants. cyanogenesis in T repens in southern France it (b) Temperature: Frost causes large ice cracks was found that different phenotypes can coexist among the cells of leaves. When the leaves simultaneously on the same plant. This report thaw the cyanoglucosides and the linamarase examines the incidence and heritability of this in a cyanogenic plant mix and HCN is within-plant variation and discusses the role of released. There is some evidence of an associ- natural selection in maintaining the genetic and ation between the frequency of the cyanogenic phenotypic polymorphisms. form and temperature. A study by Daday (1954a) on samples of white clover collected all over Europe shows a clear relationship MATERIALSAND METHODS between the January mean temperature and Foursites at different altitudes in southern France, frequencies of the dominant alleles at the two between Montpellier and the Cevennes, were loci. The association is so pronounced that a chosen for study (table 1). At each site 50 stolons 1°F (0.56°C) decrease in mean January tem- were collected. To minimize the possibility of perature corresponds to a reduction of 4-23 sampling the same genet twice the distance per cent in the frequency of the Ac allele. between successive samples was at least 1 m. The Moreover, there is a strong correlation stolons were planted in individual cubical pots between the allele frequencies at the two loci. (lOxlOxlOcm) in the glasshouse and were Other evidence shows a negative relationship allowed to root. between the frequencies of the Ac and Li The compost used was a mixture of equal parts alleles and altitude (Daday, 1954b; De of humus (Sedagri), brown peat and soil from Arraujo, 1976). (In order to calculate the allele Camp Redon (Montpellier, clayey-silt) sterilised frequencies Daday (1954a, b; 1958) assumed for 20 minutes. The glasshouse was heated in the populations to be at Hardy-Weinberg winter to keep the temperature above 15°C and equilibrium. No evidence was offered to justify cooled in summer to keep the temperature below this assumption). 27°C. No supplementary lighting was used. Thus, there are two opposing selective forces The cyanogenic phenotype of the leaves was acting on the genetic polymorphism. Neither, determined either by the modified Guignard however, appears to distinguish between the 0-, picrate paper test (Corkill, 1940), or, using the -E and --plants.The models of theoretical popu- same technique, by the Feigl and Anger (1966) lation genetics show that these selective forces reagent as adapted for plants by Tantisewie et a!. could, when acting together, maintain a genetic (1969). All leaves tested were sampled at the same polymorphism at one locus, the other locus being stage (folded leaves). Linamarase was isolated monomorphic for the dominant allele, but are from the seeds of linseed (Linurn usitatissimum L.) insufficient to explain either the polymorphism at according to Coop (1940); linamarin was obtained both loci or the regularity of the dine in Europe commercially (Cal. Biochem.). corresponding to the January mean temperature. (a) All plants collected were first tested using the Thus the continued presence of the three modified Guignard picrate test. Three or four acyanogenic phenotypes (G-, -E and --)inmost additional tests were made at various time populations must result from other factors or intervals using the Feigl and Anger reagent.

Table 1The frequency of cyanogenic Trtb1iumrepens atfour sites located in southern France, between Montpetlier and theCevennes.The proportions are based on samples of 50 individuals per site

% Site Altitude CodeLocation Cyanogenic Acyanogenic

St Mathieu 120 m SM plain 77 23 Ganges 170 m GA valley 80 20 Le Coulet 600m CO top of slope 15 85 Col du Pas 830 m CP top of slope 4 96 CYANOGENESIS IN WHITE CLOVER 267

(b) Two plants from one of the sites were studied RESULTS in further detail. A cutting was taken from each plant and established in the centre of a Fouror five repeated tests were made on individual circular shallow pot of 50 cm radius. The plants in 1981 and 1982 and showed that different growth of the stolons was mapped. When the phenotypes can coexist simultaneously in the same plants were large enough, every terminal leaf plant. That is, some leaves on a plant could be of each stolon was collected and tested for cyanogenic whereas others, on the same plant, cyanogenesis within the same hour period would contain only the cyanoglucosides, only the using the Feigl and Anger reagent. The posi- enzyme, or neither. Thus some plants are tion from which each leaf was taken was heterogeneous in the expression of their genotype recorded on the map of the plant. for cyanogenesis, a phenomenon that has been (c) Crosses were made on pairs of plants isolated called "intra-individual heterogeneity" (Till, in insect-proof cages. Pollination was done 1983). manually. The keel and the wing petals were In heterogeneous plants, one type is usually removed using small forceps; this usually more frequent than the others. There was a high removes the stamens (but not invariably) and proportion of heterogeneous plants at the two uncovers the stigma. The latter was brushed sites at high altitudes, CO and CP (600 m and with the stamens of the male donor. Each 800 m) where few plants were cyanogenic (table flower was pollinated on every second day 1). Moreover, the intensity of the cyanogenic reac- during the receptive period (5to6 days). Ten tion was generally weak at these two sites. At the inflorescences per plant were pollinated. lowland sites, GA and SM, on the other hand the Flowers were not systematically emasculated intensity of the reaction was stronger and the because T.repenshas an S-allele incompatibil- majority of plants were cyanogenic (table 1) and ity system and is practically self-sterile consistently so. (Davies, 1963). Each plant was used as both To evaluate the magnitude of these variations male and female parent and the seeds from two plants were studied in detail in the spring of the reciprocal crosses were harvested separ- 1982. One of them was homogeneous GE and the ately. Sixty seeds per cross were scarified by other was heterogeneous, predominantly -E. Both hand and left to germinate on filter paper in were chosen from the GA sample because these petri dishes. Successful germination ranged plants gave a strong reaction in the test. The from between 25and55seedsper cross. As homogeneous plant was tested twice and the soon as the radicle appeared, the seedlings heterogeneous one on 5separateoccasions. The were planted in individual pots. In order to results are shown in table 2. evaluate heterogeneity ten leaves from each Even the homogeneous plant (GE) was not offspring were tested individually with the absolutely consistent (4 samples out of 141 were Feigl and Anger reagent for their cyanogenic acyanogenic) when studied in this detail and the phenotype within the same hour period. Only HCN released was greater by peripheral than by the plants that gave the same result for all 10 central leaves. In contrast, the heterogeneous plant leaves are regarded as homogeneous; all the showed great variation in the expression of both others show intra-individual heterogeneity. G and E (39 samples out of 274). Moreover, the

Table 2Cyanotype of the terminal leaves of two plants at different dates. One plant was homogeneous, GE,theother was heterogeneous, predominantly -E. All the terminal leaves of the plants were collected and tested for their cyanogenic type within the same hour

Number of leaves Date GE G- -E -- Total

Homogeneous 3 May 83 60 1 1 0 62 18May83 77 0 2 0 79 Heterogeneous 2 May 83 10 3 27 3 43 19May83 6 1 58 0 65 9June83 2 0 50 0 52 23June83 2 0 58 0 60 22July83 4 0 42 8 54 268 I. TILL heterogeneity was greater for the glucosides than heterogeneity increases with the number of for the enzyme (p

Table 3Distribution of the offspring of crosses between homogeneous (Ho) and heterogeneous (He) plants according to their heterogeneity Number of offspring Heterogeneous Parents Homogeneous 1 2 3_5* Total

HotxHo2 43 7 0 1 8 J-iolxHe2 39 7 1 0 8 Hoix He! 29 6 5 5 16 He3xHe2 17 11 7 7 25

*Thenumbers indicate the number of samples, out of 10, per plant, that gave a different result than the predominant cyanotype. All crosses were made with Ho and He both as maternal and paternal parents. As no differences were found between the progeny from the reciprocal crosses the data have been combined. CYANOGENESIS IN WHITE CLOVER 269

Table 4 Observed (o) andexpected(e) values for each function (G or E) for four crosses between homogeneous (Ho) and heterogeneous (He) plants. The expected values are calculated according to the two loci two alleles model. The hypothetical genotype of the parents is noted for each cross

Offspring Parents G - E -

Hol (GE) x Ho2 (GE) o 33 18 47 4* AcacLj!i AcacLili e 3825 1275 3825 1275 Hol (GE) x He! (G-) o 35 10 26 19 AcacLili Acacli0 e 3375 1125 2250 2250 Ho! (GE) x He2 (G-) o 44 3* 27 20 AcacLili Acaclili e 3525 !!75 2350 2350 He3 (G—) x He2 (G—) o 42 0 0 42 AcAclili Acaclili e 42 0 0 42

* p

1963), but these have either been overlooked or probably means that intra-individual heterogeneity attributed to modifier genes segregating in these has been eliminated by human selection. When particular families (Atwood and Sullivan, 1943). intra-individual heterogeneity has been observed Aberrant progenies have also been recorded in the successive samples have usually been taken at formal breeding studies with L. corniculatus (Ram- different times of the year. Heterogeneity was nani and Jones, 1984) even though an independent therefore observed in time, but not in space and genetic marker (colour of the keel petals) segre- attributed to flexibility in the expression of the gated normally in these same progenies. genotype related to temperature or season (e.g., In my experiment, two crosses out of four gave with L. corniculatus, Ellis et a!. 1977). aberrant ratios and suggested multigenic inheri- Only rarely have repeated samples been taken tance. Moreover, by testing different leaves of the from the same individuals. More usually people same age from the same plant at the same time, I have tested only one sample of leaves from each have found that a plant can show simultaneously individual plant and could not, therefore, have two or more possible phenotypes for cyanogenesis, detected intra-individual heterogeneity. However, a phenomenon termed intra-individual this intra-individual heterogeneity of phenotype heterogeneity (Till, 1983). means that the presence of different cyanogenic In the literature on cyanogenesis the stability phenotypes in a population may neither corre- of the phenotype is usually not contested (except spond with nor result from genetic diversity at the for Ellis et al., 1977; Ramnani and Jones, 1984 Ac and Li loci. At the extreme, intra-individual with L. corniculatus), and is even sometimes asser- heterogeneity could account for what appears to ted. Ware (1925), for example, wrote "not only be a polymorphism at both loci even though all was the property of cyanogenesis constant from individuals in the population could be genotypi- one year to another, but so also, to a marked cally Ac Li. We now have two interrelated prob- degree, was the intensity of the colour change". lems. The breeding evidence shows that there is a (He was using the sodium picrate test on T repens.) genuine genetic polymorphism for cyanogenesis In contrast, Rogers and Frykolm (1937) found in T repens, while at the same time the genetic individual plants of T. repens that could give both polymorphism is being mimicked by a phenotypic negative and positive reactions when tested for polymorphism. Are the same selective factors their ability to release HCN. Williams (1939) also involved in the two situations? observed that "duplicate tests sometimes gave con- The intra-individual heterogeneity means that flicting results". De Waal (1942) observed diurnal the frequency of cyanogenesis in natural popula- variation within plants. Generally, no within-plant tions does not really refer to populations of plants, variation is mentioned when the authors have but rather to populations of leaves. We have, there- worked with plants grown from commercially fore, populations of leaves of T. repens in which available seeds (e.g., Corkill, 1940, 1942; Maher the frequency of the cyanogenic form is positively and Hughes, 1973). The authors who worked with correlated with the January isotherms in Europe wild plants (Rogers and Frykolm, 1937; Atwood (Daday 1954a), or negatively with altitude and Sullivan, 1943) found some variation. This (Daday, 1954b; De Arraujo 1976). The plants used 270 I. TILL in those experiments were obtained as seeds or proposed is a totally randomised one where the cuttings from natural habitats. They were grown strategy of the organism is unpredictable. In the up and tested in a glasshouse and therefore all case I have described, individual white clover plants were in essentially the same environment plants do not seem to have a deterministic strategy when tested. Thus what was observed was not a as no pattern of variation can be detected in time measure of the frequency of the cyanogenic or in space. It appears that for each plant the phenotypes in situ, but probably a more direct cyanotype of a leaf is determined at random. Con- effect of the underlying genotype of the plants. sequently, where there are differences between The intra-individual heterogeneity isless leaves within the same plant and between plants marked in plants that would be scored as GE than there is a cyanogenesis mosaic in the field that is in the other three phenotypes (Till, 1983). These highly diversified both in space and in time. This GE plants are relatively stable irrespective of alti- strategy is not favourable to herbivores as they tude although they are less strongly cyanogenic at cannot predict the cyanotype of a leaf from the higher altitudes. Furthermore GE plants are less previous one eaten or sampled. common at higher altitudes. This suggests that T repens is a species with very great genetic there is a positive response by the plant to selection variability and plasticity for growth and flowering against the GE phenotype in addition to the totally characters (Burdon, 1980). Turkington eta!. (1979) passive one of GE plants being eliminated by selec- have demonstrated variation in agressiveness tion. At some locations, selection appears to have towards a standard clone and found a great diver- favoured plants that are capable of changing sity of types at the local scale in the field. The phenotype in response to some environmental cue. plant also shows great diversity in the pattern of This situation is similar to the one described by markings on the leaves (Carnaham et a!., 1955; Jones and Ramnani (1985) in L. corniculatus, but Cahn and Harper,1976).Intra-individual whereas they were discussing changes of cyanogenic heterogeneity is another element phenotype with time the situation in individuals showing the extreme intra-specific diversity both of T. repens concerns differences of phenotype in between and within individuals of T. repens L. space. The outcome may well be the same in that the frequency of cyanogenic plants/leaves in a AcknowledgementsThiswork was part of a collaborative population can change without the elimination of programme on the "Polymorphism in white clover" and sup- any individuals (see Jones and Ramnani, 1985). ported by grants from: Centre National de Ia Recherche This type of "phenotypic plasticity" is very flexible Scientiliquc A1P Internationale 1981), Scientific Research Council (France-UK Collaborative Study, RDI. 54), and and economical because the substances involved NATO (Research Grant 032-81). 1 wish to thank Professor G. can be recovered, whereas morphological changes Valdeyron, Drs P. H. Gouyon and P. Jacquard for their help are fixed. and advice throughout this work. Assistance in experimental Intra-individual heterogeneity can be observed work was provided by M. Heltran and the stall of the CEPF. at two levels. Two leaves from the same plant and experimental station. 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