Genetica (1972) 43: 394-406

ON THE POLYMORPHISM OF CYANOGENESIS

IN LOTUS CORNICULATUS L. IV. THE NETHERLANDS

DAVID A. JONES Department of Genetics, University of Birmingham, B15 2TT, England

Received March 20, Ig72/Accafited April 20, 1972

A continuous gradual increase in the frequency of the cyanogenic form of Lotus corniculatus L. from 3 per cent near Den Helder to 96 per cent in Goeree has been found on the high dunes of west Holland. It is possible that grazing by rabbits may have contributed to the origin and maintenance of the cline.

Introduction

On the high dunes in the western part of the Netherlands, Lotus corniculatus L. is an extremely common plant. Studies which began in 1969 have revealed a clear pattern in the distribution of the cyanogenic form of the plant on these dunes. In other parts of the Netherlands the plant is much rarer and, because of the efficient use of land, it is often difficult to find more than a dozen plants in any one location. Large groups of plants can, however, be found in some habitats, but with- out the help of local field botanists the searching can be very tedious.

Previous work has indicated that differential eating of the acyano- genie form of the plant by various animals (JONES, 1966; CRAWFORD- SIDEBOTHAM, 1972) and the disadvantage experienced by the cyano- genie form in droughting conditions (FOULDS & GRIME, 1972) coultl well have an effect on the polymorphism of cyanogenesis, but as yet there is little direct evidence (JONES, 1962) that the former process is of importance in the wild. The study of L. corniculatus in the Nether- lands does suggest that selective grazing occurs on the dunes, but it must be admitted that the evidence is only circumstantial.

Materials, Methods and Results

The techniques used for testing the plants and the genetic systems

POLYMORPHISM OF CYANOGENESIS IN LOTUS 395

TABLE 1

PHENOTYPES OF LOTUS CORNICULATUS WITH RESPECT TO CYANOGENIC

GLUCOSIDE AND @-GLUCOSIDASE PRODUCTION

Plant Contains

Shorthand Notation,

Gross Phenotype Glucoside first

glucosides and enzyme cyanogenic glucosides, no enzyme acyanogenic enzyme, no glucosides acyanogenic neither glucosides nor enzyme acyanogenic

++ i- - - + Not distinguished - -in this study

involved have been described elsewhere (JONES, 1966). No attempt, however, was made to distinguish between the two types of plant which do not contain the cyanogenic glucosides linamarin and lotaustralin. Thus only two of the four possible phenotypes (Tab. 1) were scored unambiguously. The precise genotypes of individual plants were not determined, but the tetrasomic mode of inheritance of the glucosides has been described by DAWSON (1941) while BANSAL (1966) has re- ported on the inheritance of the P-glucosidase.

Systematic plant sampling was used wherever possible and, because of the ability of the plant to clone, no leaf sample was taken within one metre of the previous one. On the other hand, there is a genetical polymorphism of brown tip as opposed to yellow tip of the keel petals (HART & WILSIE, 1959) and when the plants are flowering this charac- ter can be used to distinguish individuals growing close together.

Data collected over the three years 1969, 1970 and 197 1 are pre- sented in Tables 2 and 3. A total of 20 12 plants have been included, of which 1020 were located on the dunes and dykes of the west coast. Tab- le 4 shows the analysis of variance of regression of the frequency of the cyanogenic form on the map reference northing, using only the data from Table 2. The most obvious conclusion to be drawn from Table 2 is that the distribution of the cyanogenic form in the west of the country is clinal, with an increase from almost zero per cent to almost 100 per cent over a distance of 134 km (i.e. approx. 0.75 per cent per km). Presenting the data in a different form the graphs in Figure 1 show the frequencies of the cyanogenic and the glucoside containing

TABL

E 2

THE

SAM

PLES

O

F LO

TUS

CO

RN

ICU

LATU

S FR

OM

H

ABIT

ATS

ON

THE

DU

NES

AN

D

DY

KE

S

OF

WES

TER

N

HO

LLAN

D

Loca

lity

No.

of pla

nts

ot Pe

rcent-

Pe

rcent-

Ma

p Pe

rcent-

ea

ch

phen

otype

ag

e as

re

feren

ce

age

++

+- -+/

-- ++

t?

North

ing

CaCO

s( 1)

km

Mean

wi

nter

from

rainf

all

in 2 m

Huis-

mm

(2)

duine

n

Den

Helde

r Gr

oote

Keet

en

Calla

ntsoo

g Pe

tten

(3)

Harg

en

Berg

en

1 2 3 Eg

mond

Bi

nnen

Ca

stricu

m W

ij k

aan

Zee

Bloe

mend

aal

Noor

dwijk

1 2

Katw

ij k

Groo

te Be

rkheid

e Zw

arte

Pan

Was

sena

ar ‘

Kij

kduin

20

) Mo

nster

Go

eree

2 0 0 1 6 3 0 0 2 14 7 8 8 a 22

24

62

50

12

9 39

25

2 29

7

46

4 71

0

3 3

57

20

24

17

30

8 17

5

45

3 45

11

25

7

36

13

29

2 25

6

36

6 22

8

18

7 9

10

3 7

0 4

10

12

0 1

0

3.1

9.4

5500

3.6

16

.4 54

43

0 5.3

53

79

0 0

1.7

6.5

5240

12

.0 54

.0 51

99

6.0

40.0

5187

0

32.0

5176

I

0 10

.0 51

23

4.0

10.0

5069

28

.0 50

.0 50

14

14.0

28.0

4909

16

.0 42

.0 47

43

22.9

28.6

4720

16

.0 28

.0 46

75

44.0

56.0

4648

48

.0 64

.0 46

44

79.5

88.5

4640

79

.3 95

.2 46

29

63.2

100.

0 45

52

76.5

100.

0 45

19

96.1

100.

0 42

82

0.6

0.2-0

.9

1.9-2

.3 2.7

4.3

3.3

4.0

2.0

4.1

2 8 11

22

26

32

33

34

40

46

51

72

a0

83

88

91

91

92

93

107

108

109

136

191.

8

175.

0

158.

4

175.

9

z E;

196.

4 18

6.9

9

197.

4 “0

182.

2

201.

5

204.

6

192.

4 17

7.2

1 Pe

rcenta

ge

CaCO

s fro

m DO

ING

(196

6).

2 Fr

om

Klim

atolo

gisch

e Ge

geve

ns

( 196

9).

3 Yo

ung

plants

on

dis

turbe

d lan

d.

POLYMORPHISM OF CYANOGENESIS IN LOTUS 397

TABLE 3

THE SAMPLES OF LOTUS CORNICVLATUS FROM HABITATS OTHER THAN ON THE

DUNES AND DYKES OP WESTERN HOLLAND

Locality Sneek (Roadside) Kleine Sluis

(Sandy ridge) Oude Mirdum (Klif) Hoorn (Roadside) Joppe (Roadside) East of Leiden

(Roadside) Veenendaal

(Sandy ridge) Drempt (Field)

(Roadside) Nr. Heelsum (Fields) Nr. Wageningen

(Fields) Slijk Ewijk

(Waal Bank) Zaltbommel

(Waal Dyke) Beers (Roadside) Odiliapeel

(Roadside)

No. of plants of each Per- phenotype centage Map

- ++ +- -+/-- ++

29 0 0 100

5 9 45 8.5 0 0 159 0 2 6 33 4.9

32 9 9 64.0

11 7 39 19.3

0 2 49 0 44 3 10 77.2

3 5 12 15.0 31 2 0 93.9

65 1 1 97.0

54 0 1 98.2

103 0 0 100 45 3 2 90.0

26 3 12 ’ 63.4

reference 1722i5612

1164 5409 1640 5390 1314 5187 2120 4787

0972 4653

1647 4503 2059 4486

1825 4421

1703 4406

1816 4330

1479 4242 1840 4160

1793 4082

TABLE 4

ANALYSIS OF VARIANCE OF REGRESSION OF THE FREQUENCY OF THE CYANOGENIC

FORM OF LOTUS CORNICULATUS ON THE MAP REFERENCE NORTHING

d.f. MS V.R

regression 1 7861.970 58.31 remainder 19 134.830 P <O.OOl

Angular transformation of the frequency data was used.

DAVID A. JONES

n 25 50 75 100 125

DISTANCE (km) from H~JISDUINEN

Fig. 1. The frequency distribution of the cyanogenic (continuous line) and the glucosidic (interrupted line) forms of Lotus corniculatus on the high dunes of

west Holland. Data in Table 2.

folms plotted against distance form Huisduinen. Nowhere in the cline is the allele determining the cyanogenic glucosides absent and there are several places where marked steps in the distribution occur.

Discussion

It is clear from Table 2 that the sample sizes are small. If the dif- ferences observed had been trivial, then criticism of the small numbers of plants tested at any one locality would be justified. But the differ- ences between frequencies in samples from different places are so large and the cline is so spectacular that it must be concluded that the differ- ences observed are real and meaningful. The data in Table 5, moreover, show that there is consistency between samples collected in the same locations in different years. This is true for both of the sites in the Netherlands which were sampled twice and for other locations in Denmark and in the British Isles. The data also suggest that these

POLYMORPHISM OF CYANOGENESIS IN LOTUS 399

TABLE 5

DUPLICATE SAMPLES OF GROUPS OF LOTUS CORNICULATUS PLANTS TAKEN IN

DIFFERENT YEARS

Locality Year ++ f- -+/-- N %

++

Porthdafach

(N. Wales)

Rounds Green

(Staffs, England)

Sundstrup

(Jutland, Denmark)

Callantsoog 1969

(Noord-Holland) 1970

Wassenaar 1970 31

(Zuid-Holland) 1971 62

1959 5

1966 14

1970 14

1967

79

88

3

5

0

0

15 20 25.0

36 51 27.5

a 29 50 28.0

4 83 95.2

12 100 88.0

8 a3 94 3.2

23 144 172 2.9

5 45 50 0

4 46 50 0

7 3 41 75.6

7 9 78 79.5

differences between the frequencies in different localities are likely to be stable.

A major disappointment during the field work of 1969 and 1970 in the Netherlands was the small number of locations in which L. corni- culatus was found between the high dunes and I Jsselmeer. More time was spent searching for plants on the polder than was taken testing the plants on the dunes and yet few were found. Lotus uliginosus Schkuhr is common in the low lying, wet regions, but L. corniculatus appears to be a relatively rare species there. Having established the existence of the cline in 1970 it became necessary to determine wheth- er this cline was an effect of latitude or a characteristic only of the dunes. With the help of Mr. R. M. van Urk, the specimens stored in the Rijksherbarium, Leiden were searched systematically in 197 1 and the sites of plants collected from the polder were recorded. It became ap- parent that the most common habitats on the polder were cemeteries (begraafplaatsen), raised above the polder, but the number of plants found in any one cemetery was too small to sample sensibly.

The nine new locations sampled in 1971 do not help to solve the

400 DAVID A. JONES

problem of the cline because only three of them are inland from the dunes of Noord and Zuid-Holland. Furthermore, the frequency of the cyanogenic forms at Kleine Sluis, Hoorn, and east of Leiden are in no way exceptional in relation to the frequency on the dunes at compara- ble latitudes. This may indicate a latitudinal effect, but there is no independent evidence available at present.

The groups of plants at Veenendaal and at Seek are exceptional in their respective areas. The twenty-nine plants tested near Seek were similar in morphology to others found by the sides of new roads in Denmark and England. It is likely that these plants are alien con- taminants of the grass seed used to sow the roadside verges. At the Veenendaal location, the soil is fine poor sand (Gley) and is very dif- ferent from the surrounding soils. A canal cuts through a ridge at this place and the management of the land is much less strict than in neighbouring areas. Other samples in the immediate vicinity need to be collected.

THE CLINE AND HABITAT FACTORS

The BODEMKAART VAN NEDERLAND (1961) and the ATLAS VAN NEDERLAND (1963) show that from Den Helder to Bergen aan Zee the basis of the soil is non-loamy fine sand with a low humus content. The dunes to the south have essentially the same soil type except for being slightly calcareous. The climatic conditions are remarkably uniform along the whole coastal strip. The first and last frosts occur at the same time; the yearly mean temperature is the same; the number of frosty days in which the temperature does not rise above 0°C is 8 to 9 at Goeree rising to 10 to 11 at Den Helder. Along the whole coast, except for Den Helder, there are 10 to 15 days in the year during which the temperature reaches at least 25°C.

There is scarcely more than a 1 “C difference in the mean temperature in any month over the whole area, while for rainfall it appears to be wetter to the south (Tab. 2). On the other hand, there is no correlation between the frequency of cyanogenic plants and rainfall (Tab. 6) even when data most favourable to a correlation are selected from the climatological records.

As far as the vegetation is concerned, it is a matter of common ob- servation that the dunes in the north are unstable and Ammofihila

POLYMORPHISM OF CYANOGENESIS IN LOTUS 401

TABLE 6

ANALYSISOFVARIANCEOFREGRESSIONOFTHEFREQUENCYOFTHECYANOGENIC

FORM ON THE MEAN WINTER RAINFALL OVER THE YEARS 1951-1960 (Data in Table 2)

d.f. MS VR

Regression 1 1107.472 1.81 Remainder 10 611.576 P >0.20

arenaria has been planted over large areas. On travelling south it is apparent that the dunes are more stable, shrubs occur with increasing frequency and there is a marked increase in the number of grass species. The vegetation in the southern regions is, therefore, less arti- ficial.

Detailed analyses of two areas covered by this work have been des- cribed by DOING (1963, 1966). These concern the regions between IJmuiden and Noordwijk to the south and between IJmuiden and Camperduin to the north, but unfortunately neither of these concern the region of particular interest in the present study, viz. between Noordwijk and Scheveningen. DOING (1966) has, however, listed the calcium carbonate (shell fragments) content of soil samples collected on the dunes (cf. Tab. 2) and thereis asteadyincrease from Camperduin to Voorne. This clearly parallels the cline, but as yet no effect of CaCOs content of soils on the polymorphism of cyanogenesis has been shown in any formal manner. This must now be investigated. CORKILL (1952)

has described preferential grazing of acyanogenic Trifolium repens by rabbits, and JONES (1966) demonstrated selective eating of acyanoge- nit L. corniculatus by voles. Although there are some exceptions, CRAW-

FORD-SIDEBOTHAM (1972) has shown that most of the molluscs whicheat acyanogenic T. repens differentially also behave in the same way with L. corniculatus. Thus there is reasonable experimental evidence to indicate that there may be a relationship between the presence of these animals and a high frequency of cyanogenic plants in natural populations. Little evidence of rabbit faeces was found at Egmond Binnen, Noordwijk and Katwijk in 197 1, but pellets were so common at Wassenaar that three counts gave 5, 9 and 14 pellets per 10-i ms res- pectively.

402 DAVID A.JONES

The presence of rabbits can also be inferred from their influence onthepatternofvegetation (VANLEEUWEN&VANDERMAAREL, 1971) and the photographs in Figures 2 and 3, taken at Egmond Binnen and Wassenaar in July 1971 demonstrate this clearly. When rabbits are rare or absent, there is a gradual fall in the height of the vegetation from the scrub to the short turf. Rabbits graze close up to the scrub and their presence can be detected by the sharp cut off in the height of the vegetation at the edge of the scrub. This is very marked in Fig. 3. Rabbit grazing, therefore, is an important difference between the habitats at Egmond Binnen, Noordwijk and Katwijk on the one hand, and Wassenaar on the other and at the latter place only is there a high frequency of cyanogenic plants. This field evidence, together with the experimental evidence outlined above, suggests that rabbit grazing may well be an important factor in determining the distribution of cyanogenic L. corniculatus on the high dunes. Certainly there is a prima facie case for examining the populations further. The relation- ship between the presence of rabbits and a high frequency of cyanoge- nit plants could be verified by studying the effect of the spread of myxomatosis-resistant rabbits back to their old haunts where at pre- sent the acyanogenic forms of L. cornicdatus appears to be at an ad- vantage.

In his discussion of the evolution of morph-ratio clines, CLARKE ( 1966) developed models which suggest that over a period of time there will be a trend towards the steepening of a cline and towards the for- mation of one or more sharp steps within it. The graphs in Figure I show distinctly that the cline on the coastal dunes is broken up into steps. The most noticeable ones are between Castricum and Wijk aan Zee and between Katwijk and Wassenaar. North of Castricum and south of Wassenaar the frequencies are relatively stable.

It is the region between Katwijk and the road from Wassenaar to Wassenaarse Slag which is the most critical. There is an increase in the frequency of the cyanogenic form from approximately 20 per cent to almost 80 per cent over a distance of less than 5 km. The samples at Groote Berkheide and Zwarte Pan are almost midway between these frequency extremes. Further studies of the plants and the habitats south west of Leiden will be needed to determine whether there are any particular habitat factors (i.e. rabbits) influencing the polymorphism on the dunes in that region.

POLYMORPHISM O F CYANOGENESIS I N LOTUS 403

Fig. 2. The vegetation at the Egmond Rinnen habitat.

Fig. 3. The vegetation a t the Wassenaar habitat.

404 DAVID A. JONES

TABLE 7

MAJOR DIFFERENCES IN THE FREQUENCY OF THE CYANOGENIC FORM OF LOTUS CORNICULATUS IN ADJACENT GROUPS OF PLANTS

++ N ++ N Year

Silbury Hill 2 39 10 28 1958

(Wilts) South Face North Face Porthdafach 14 51 42 74 1970 (Anglesey Sea Cliff Inland from Cliff Ballidon 29 36 17 40 1971 (Derbyshire) Poorly drained soil Well drained soil Trearddur Bay 5 39 21 39 1959 (Anglesey) SE, W, S facing slope NW facing slope.

None of the models suggested by HALDANE (1948), FISHER (1950) and CLARKE (1966) are strictly relevant to tetraploid perennial plants. With cyanogenesis in Lotus corniculatus there is, moreover, comple- mentary gene interaction, because cyanide production requires the presence both of the glucosides and of the enzyme, each being pro- duced by a dominant allele at unlinked loci. The most helpful dis- cussions are by PARSONS (1959) and MAYO (197I), but even these assume duplex (GGgg) advantage. At present, therefore, it appears that there is no adequate algebra for clinal situations in autotetra- ploids.

The work on cyanogenesis shows that clines occur at three levels. (1) The overall distribution in Europe of the cyanogenic form of T. repens (DADAY 1954) shows an increase in the frequency from the north east to the south west. With Lotus corniculatw the effect is similar but not as spectacular (JONES, in preparation). (2) At the mid-level there is the cline on the coastal dunes of the Netherlands associated with latitude, soil calcium content and probably with rabbit grazing. (3) Changes in frequency over very short distances also occur (Tab. 7). Although there are clear and complex differences between the adjacent habitats at Silbury Hill, Porthdafach and Trearddur Bay, they were not noted before the samples were taken. The adjacent groups of plants at Ballidon were chosen because of a marked difference in soil drainage (ephemeral plants were used as an indicator) and so the dif- ference obtained can be regarded as a test of the hypothesis of FOULDS & GRIME (1972).

POLYMORPHISM OF CYANOGENESIS IN LOTUS 405

Thus, there is an overall strategy in the distribution of the cyanoge- nit forms associated with temperature while the tactics is related to soil water, grazing and other discontinuities of the local habitat.

I am most grateful to the Science Research Council (B/SR/6494) for the fi- nancial support of this work. It is a pleasure to acknowledge the help and advice of Dr. HENK DOING, Dr. A. J. VAN DER LINDEN, Drs. J. H. WIEFFERING, Mr. D. T. E. VAN DER PLOEG and Mr. R. M. VAN URK, whose knowledge of the Flora and Ecology of the Netherlands has enabled me to find plants in unexpected habitats. I also thank Professor Dr. R. HEGNAUER and Drs. J. MENNEMA for hospitality in their respective laboratories and Dr. T. J. CRAWFORD-SIDEBOTHAM for his help with the field work in 1969.

REFERENCES

ATLAS VAN NEDERLAND (1963) Staatsdrukkerij- en Uitgeverijbedrijf, ‘s-Gravenhage.

BANSAL, R. D. (1966). Studies on procedures for combining clones of birdsfoot trefoil, Lotus corniculatus L. Thesis, Cornell University.

BODEMKAART VAN NEDERLAND (196 1). Stichting voor bodemkartering. Wage- ningen

CLARKE, B. C. (1966). The evolution ot morph-ratio clines. Am. Nat. 100: 389- 402.

CORKILL, L. 11952). Cyanogenesis in white clover (Trio/o&m repens L.) VI. Experiments with high-glucoside and glucoside-free strains. N.Z. JZ Ski. Tech., (A), 34: l-16.

CRAWFORD-SIDEBOTHAM, T. J. (1972). The role of slugs and snails in the mainte- nance of the cyanogenesis polymorphisms of Lotus corniculatus and Trifolium repens. Heredity 28: 405-4 11.

DADAY, H. (1954). Gene frequencies in wild populations of Trifolium repens. I. Distribution by latitude. Heredity 8: 61-78.

DAWSON, C. D. R. (1941). Tetrasomic inheritance in Lotus corniculatus L. J. Genet. 42 : 49-72.

DOING, H. (1966) Beschrijving van de vegetatie der duinen tussen I Jmuiden en Camperduin. Meded. Landb. Hoogesch. Wageningen, 66 (13) : l-63.

DOING KRAFT, H. (1963). Eine Landschaftskartierung auf vegetationskundlicher Grundlage im Masstab 1: 25000 in den Diinen bei Haarlem. In: R. T~~XEN (Ed), Bericht iiber das Internationale Symposium fiir Vegetationskartie- rung 1959 in Stolzenau/Weser, pp.: 297-312. Belnzontia (II) Ecology, fast. 10.

FISHER, R. A. (1950). Gene frequencies in a cline determined by selection and diffusion. Biometrics 6 : 353-36 1,

FOULIX, W. & GRIME, J. P. (1972). The influence of soil mosture on the frequen-

406 DAVID A. JONES

cy of cyanogenic plants in populations of Trifoliuln repens and Lotus covni- culatus. Heredity 28 : 143-146.

HALDANE, J. B. S. (1948). The theory of a cline. J. Genet. 48: 277-284. HART, R. H. and WILSIE, C. P. (1959) Inheritance of a flower character, brown

keel tip, in Lotus corniculatus L . Agron. J . 51 : 379-380. JONES, D. A. (1962). Selective eating of the acyanogenic form of the plant

Lotus covniculatus L. by various animals. Natuve 193: 1 109-1 110. JONES, D. A. (1966). On the polymorphism of cyanogenesis in Lotus covniculatus.

Selection by animals. Can. J. Genet. Cytol. 8 : 556-567. KI~IMATOLOGISCHE GEGEVENS VAN NEDERLANDSE STATIONS (1969). NO. 2. Aver-

ages for the period 1951-1960. Royal Netherlands Meteorological Institute, De Bilt.

LEEUWEN, C. G. VAN & E. VAN DER MAAREL 11971). Pattern and Process in Coastal Dune Vegetations. Acta. Bot. Neerl. 20 : 191-198.

MAYO, 0. (1971). Rates of change in gene frequency in tetrasomic organisms. Genetica 42 : 329-337.

PARSONS. P. A. (1959) Equilibria in auto-tetraploids under natural selection for a simplified model of viabilities. Biomet~ ics 15: 20-29.