Ecological genetics of Trifolium repens

The theory of Evolution by Natural Selection deals substantially with events now past, and with processes too slow for contemporary study. Experimental studies of evolution are possible, but are liable to the criticism that they deal with artificial, or at least extreme and atypical situations, involving rapid evolutionary change. Such criticism can be avoided to a degree by studying situations now in equilibrium.
It is suggested that interesting results may be obtained by studying polymorphisms, and comparing the equilibria reached in populations of an organism in different environments. The chosen approach was to find a case of polymorphism convenient to study, followed by an attempt to interpret the pattern of its variation in terms of features of environments where a morph was common selecting in its favour, and vice versa.
The material presented falls into three parts.
1) Material and Methods: Background and Preliminary Studies.
The system chosen for study was the white V-shaped leaf markings of Trifolium repens. The species is widespread, abundant, and variable; the character is expressed on vegetative organs, and is therefore always accessible; and the known variation in this character had already been interpreted in terms of the action of a set of several alleles at one locus.
The results of crosses indicate general agreement with previous reports. One heterozygous plant apparently largely failed to transmit one allele, Vb, through the pollen. Segregations indicate that a new phenotype, 'smeared', is determined by a particular Vh allele. Two more new phenotypes, 'shaded' and 'marginal' are described. The places where these and other rare phenotypes have been found are listed. New reports and the author's other experience are combined with previous reports in a new general account of leaf marking in the species, which also deals with at least three distinct classes of red markings. There appears to be more variation, implying more alleles, than previous accounts allow for, and reportedly distinct types, for example, Vh, Vl and Vf, seem to be linked by a series of intermediates, among which clear dividing lines are hard or impossible to draw.
Experimental studies relating to the techniques of population sampling are reported. Scarification by 10 to 20 minutes exposure to concentrated sulphuric acid was chosen to deal with hard seed. The effect of a restricted number of mother plants on the accuracy of a seed sample is discussed; predictions concerning the frequencies of different types in progeny from single heads grown separately were roughly consistent with observations. Possible differences between vegetative and seed samples are discussed. Morph frequencies in a sample of plants bearing inflorescences were not found to be significantly different from those of the whole vegetative sample. The consequences of this species' ability to reproduce vegetatively are discussed, and a brief clone-mapping project is described.
The procedures used to obtain and score material, and to obtain information about its background are described. The amounts of material of different types from different sources is summarised in Table 6.2. Problems involved in estimating gene frequencies are discussed, and the derivation of the values used to represent the different morph frequencies is outlined; they are basically phenotypic frequency figures, gene frequency calculations being used only where it was found to be necessary.
2) Observations on Population Samples.
Polymorphism in respect of white leaf marks was found to be present in all except 15 out of 624 samples. The commonest group of phenotypes are the 'simple V-marks', referred to as 'L'. The next commonest is the unmarked type, 'O', present in all except 72 out of 624 samples, with an overall frequency among the plants scored of 17 per cent.
Study of the sample data reveals a deficiency of the double marked phenotypes expected to be showed by VbyVl plants. This is explained as the result of a degree of dominance of Vby in such combinations; the effect of such dominance is allowed for in the frequency figures representing the frequency of 'L' marks, and of double-marks containing two members of the 'L-series' of simple-V-producing alleles.
The possibility of demonstrating interaction between the frequencies of different morphs is discussed. There are indications of lower frequencies of 'By', 'B', and 'F' marks when the unmarked phenotype is common.
The possibilities for the main object of the work, discovering associations between morph frequencies and environmental factors are shown in Table 8.1, giving the sets of data presentable for particular methods of analysis.
Data on 148 British samples, scored in the field by the author, were treated by Multiple Regression Analysis (using the KDF 9 Computer of the Oxford Computing Laboratory), to test for association of morph frequencies with geographical location, altitude, and soil pH. Significant increases in the proportion of unmarked plants are shown with greater distance north and higher altitude.
Frequency data for all the classes of morphs described is shown in the form of maps, of the British Isles, Western Europe, and the whole of the species' natural range. These maps confirm the northwards increase in frequency of the unmarked form found in Britain. Maps for the rare morphs show various patterns, most of which seem to involve central regions of higher frequency north of the Mediterranean (France - Alps - Greece) with lower frequencies elsewhere.
Regression analysis of a set of seed samples from Spain supports the conclusion from British vegetative samples of an increase in unmarked frequency with higher altitude. Examination of the information about the background of other samples suggests higher unmarked frequencies in pastures than on waste ground, meadows being intermediate.
British data suggest an association with wet, and particularly with badly-drained sites, with trodden paths, and with dense vegetation. There are patterns of response to water regime elsewhere but (e.g. Polish data) they tend to suggest the opposite association, of high unmarked frequency with dry conditions.
3) Comparison with other species.
New observations are reported on some other related species. Available information on marking in other species of Trifolium is reviewed, and it is pointed out that it is scattered, difficult of access (much unpublished), and sometimes ambiguous or contradictory.
Summary of this data indicates the presence of white V-markings in nearly 30 species of Trifolium, and its absence in at least as many more. Marks are probably entirely absent in subgenus Chronosemium and perhaps in part of subgenus Trifolium. but seem to occur in most of the other subgenera for which there is information. When marks are present, it appears that they are nearly always variable. Only three species are definitely reported as always marked, and in two of these there is variation between different types of marking. This suggests that the factors producing or preserving polymorphism in T. repens act also in other marked species. The presence of red leaf marks of various kinds is reported in 21 species of Trifolium.
Also in the tribe Trifolieae, both red and white V-markings are found in Parochetus communis. Material grown by the author showed great variability within each plant in leaf mark, but no clear differences between plants. Red leaf marks, variable, and in some cases approaching a V-shape, are also found in Medicago.
Several lines of evidence indicate relationships between the white markings, and the various red marking systems in T. repens. It is suggested that the white and red V-markings have a common evolutionary origin.
Some examples of leaf marking with analogous properties in genera unrelated to T. repens are also briefly reviewed.
In discussion some possible challenges to the validity of the results claimed are discussed, and evidence is presented suggesting that the reported genetic clines in unmarked frequency are real. Selective factors affecting unmarked frequency are tentatively suggested to be temperature and water regime. The problem of relating these to markings on leaves is discussed, and also the possibility that the phenomena of leaf marking are by-products of unknown processes, and are of no intrinsic importance. It is suggested that the inter- actions between red and white markings support the hypothesis that leaf markings themselves are of selective importance; and some possibilities as to what form this selective importance might take are mentioned.
Possibilities for further work indicated in the course of the studies presented here are discussed. They include studies on important problems of population dynamics, which affect the design of techniques for sampling for leaf markings, but in which also observations on leaf markings could be used as means to ends of wider significance.
Ways in which the methods used in the present study could be improved in a repeated study are suggested; however, it is felt that the clines observed provide a starting point for experimental work, and that this might be more rewarding than further descriptive work.