Research methods in ecology

by Frederic E. Clements

Fig. 39. Position of chloroplasts in aerial leaf (1) and submerged leaf (2) of Callitriche bifida. × 250.

176. The measurement of responses to light. Responses, such as the periodic opening and closing of stomata, which are practically the same for all leaves, are naturally not susceptible of measurement. This is also true of the transpiration produced by light, but the difficulty in this case is due to the impossibility of distinguishing between the water loss due to light and that caused by humidity and other factors. If it were possible to determine the amount of chlorophyll or glucose produced, these could be used as satisfactory measures of response. As it is, they can only be determined approximately by counting the chloroplasts or starch grains. The arrangement of the chloroplasts can not furnish the measure sought, since it does not lend itself to quantitative methods, and since the relation to light intensity is too inconstant. Hesselmann (l. c., 400) has determined the amount of carbon dioxide respired, by means of a eudiometer, and has based comparisons of sun and shade plants upon the results. As he points out, however, light has no direct connection with respiration. Although the latter increases necessarily with increased nutrition, the relation between them is so obscure, and so far from exact, that the amount of respiration can in no wise serve as a measure of the response to light. As a result of the foregoing, it is clear that no functional response is able to furnish a satisfactory measure of adjustment to light, though one or two have perhaps sufficient value to warrant their use. Indeed, structural adaptations offer a much better basis for the quantitative determination of the effects of light stimuli, as will be shown later.

In attempting to use the number of chloroplasts or starch grains as a measure of response, the study should be confined to the sun and shade forms of the same species, or, in some cases, to the forms of closely related species. The margin of error is so great and the connection with light sufficiently remote that comparisons between unrelated forms or species are almost wholly without value. It has already been stated that starch is merely the surplus carbohydrate not removed by translocation; the amount of starch, even if accurately determined, can furnish no real clue to the amount of glucose manufactured. In like manner, the number of chloroplasts can furnish little more than an approximation of the amount of chlorophyll, unless size and color are taken into account. In sun and shade ecads of the same species, the general functional relations are essentially the same, and whatever differences appear may properly be ascribed to different light intensities for the two habitats. The actual counting of chloroplasts and starch grains is a simple task. Pieces of the leaves of the two or more forms to be compared are killed and imbedded in paraffin in the usual way. To save time, the staining is done in toto. Methyl green is used for the chloroplasts and a strong solution of iodine for the starch grains. When counts are to be made of both, the leaves are first treated with iodin and then stained with the methyl green. The thickness of the microtome sections should be less than that of the palisade cells in order that the chloroplasts may appear in profile, thus facilitating the counting. The count is made for a segment 100 μ in width across the entire leaf. Two segments in different parts of the section are counted, and the result multiplied by five to give the number for a segment 1 millimeter in width. Although sun and shade leaves regularly differ in size and thickness, no correction is necessary for these. Size and thickness stand in reciprocal relation to each other in ecads, and thickness is largely an expression of the absorption of light, and hence of its intensity. In the gravel, forest, and thicket ecads of Galium boreale, counts of the chloroplasts gave the following results. The gravel form (light 1) showed 3,500 plastids in the 1–mm. segment, the forest form (light .03) possessed 1,350, and the thicket form (light .002), 1,000. In these no attention was paid to the size and form of the plastids in the different leaves, since the differences were inappreciable. When this is not the case, both factors should be taken into account. Starch grains are counted in exactly the same way. Indeed, if care is taken to collect leaves of forms to be compared, at approximately the same time on sunshiny days, a count of the chloroplasts is equivalent to a count of the starch grains in the vast majority of cases. Measurements of the size of starch grains can be made with accuracy only when the leaves are killed in the field at the same time, preferably in the afternoon. Counts of chloroplasts alone can be used as measures of response in plants that produce sugar or oil, while either chloroplasts or starch grains or both may be made the basis in starch-forming leaves.

Hesselmann (l. c., 379) has employed Sachs’s iodine test as a measure of photosynthesis. This has the advantage of permitting macroscopic examination, but the comparison of the stained leaves can give only a very general idea of the relative photosynthetic activity of two or more ecads. The iodine test is made as follows:[^[17]] fresh leaves are placed for a few minutes in boiling water, and then in 95 per cent alcohol for 2–5 minutes, in order to remove the chlorophyll and other soluble substances. The leaves are placed in the iodine solution for ½–3 hours, or until no further change in color takes place. The strength of the solution is not clearly indicated by Sachs, who says: “I used an alcoholic solution of iodin which is best made by dissolving a large quantity of iodin in strong alcohol and adding to this sufficient distilled water to give the liquid the color of dark beer.” This solution may be approximated by dissolving ⅓ gram of iodin in 100 grams of 30 per cent alcohol. The stained leaves are put in a white porcelain dish filled with distilled water, and the dish placed in the strong diffuse light of a window. The colored leaf stands out sharply against the porcelain, and the degree of coloration, and hence of starch content, is determined by the following table:

1. bright yellow or leather yellow (no starch in the chlorenchym) 2. blackish (very little starch in the chlorenchym) 3. dull black (starch abundant) 4. coal black (starch very abundant) 5. black, with metallic luster (maximum starch-content)

ADAPTATION

177. Influence of chloroplasts upon form and structure. The beginning of all modifications produced by light stimuli must be sought in the chloroplast as the sensitized unit of the protoplasm. Hence, it seems a truism to say that the number and arrangement of the chloroplasts determine the form of the cell, the tissue, and the leaf, although it has not yet been possible to demonstrate this connection conclusively by means of experiment. In spite of the lack of experimental proof, this principle is by far the best guide through the subject of adaptations to light, and in the discussion that follows, it is the fundamental hypothesis upon which all others rest. The three propositions upon which this main hypothesis is grounded are: (1) that the number of chloroplasts increases with the intensity of the light; (2) that in shaded habitats chloroplasts arrange themselves so as to increase the surface for receiving light; (3) that chloroplasts in sunny habitats place themselves in such fashion as to decrease the surface, and consequently the transpiration due to light. In these, there can be little doubt concerning the facts of number and arrangement, since they have been repeatedly verified. The purpose of epistrophe and apostrophe, however, can not yet be stated with complete certainty.

The stimulus of sunlight and of diffuse light is the same in one respect, namely, the chloroplasts respond by arranging themselves in rows or lines on the cell wall. The direct consequence of this is to polarize the cell, and its form changes from globoid to oblong. This effect is felt more or less equally by both palisade and sponge cells, but the disturbing influence of aeration has caused the polarity of the cells to be much less conspicuous in the sponge than in the palisade tissue. While the cells of both are typically polarized, however, they assume very different positions with reference to incident light. This position is directly dependent upon the arrangement of the plastids as determined by the light intensity. In consequence, palisade cells stand at right angles to the surface and parallel with the impinging rays; the sponge cells, conversely, are parallel with the epidermis and at right angles to the light ray. Some plants, especially monocotyledons, exhibit little or no polarity in the chlorenchym. As a result the leaf does not show a differentiation into sponge and palisade, and the leaves of sun and shade ecads are essentially alike in form and structure. The form of the leaf is largely determined by the chloroplasts acting through the cells that contain them. A preponderance of sponge tissue produces an extension of leaf in the direction determined by the arrangement of the plastids and the shape of the sponge cells, viz., at right angles to the light. Shade leaves are in consequence broader and thinner, and sometimes larger, than sun leaves of the same species. A preponderance of palisade likewise results in the extension of the leaf in the line of the plastids and the palisade cells, i. e., in a direction parallel with the incident ray. In accordance, sun leaves are thicker, narrower, and often smaller than shade leaves.

178. Form of leaves and stems. In outline, shade leaves are more nearly entire than sun leaves. This statement is readily verified by the comparison of sun and shade ecads, though the rule is by no means without exceptions. In the leaf prints shown in figures 14 and 15, the modification of form is well shown in Bursa and Thalictrum; in Capnoides the change is less evident, while in Achilleia and Machaeranthera lobing is more pronounced in the shade form, a fact which is, however, readily explained when other factors are taken into account. The leaf prints cited serve as more satisfactory examples of the increase of size in consequence of an increase in the surface of the shade leaf, although the leaves printed were selected solely with reference to thickness and size or outline. In all comparisons of this kind, however, the relative size and vigor of the two plants must be taken into account. This precaution is likewise necessary in the case of thickness, which should always be considered in connection with amount of surface. The relation between surface and thickness is shown by the following species, in all of which the size of the leaf is greater in the shade than in the sun. In Capnoides aureum, the thickness of the shade leaf is ½ (6 : 12) that of the sun leaf; in Galium boreale the ratio is 5 : 12, and in Allionia linearis it is 3 : 12. The ratio in Thalictrum sparsiflorum is 9 : 12, and in Machaeranthera aspera 11 : 12. The thickness of sun and shade leaves of Bursa bursa-pastoris is as 14 : 12, but this anomaly is readily explained by the size of the plants; the shade form is ten times larger than the sun form. Certain species, e. g., Erigeron speciosus, Potentilla bipinnatifida, etc., show no change in thickness and but little modification in size or outline. They furnish additional evidence of a fundamental principle in adaptation, namely that the amount of structural response is profoundly affected by the stability of the ancestral type.

The effect of diffuse light in causing stems to elongate, though known for a long time, is still unexplained. The old explanation that the plant stretches up to obtain more light seems to be based upon nothing more than the coincidence that the light comes from the direction toward which the stem grows. Later researches have shown that the stretching of the stem is due to the excessive elongation of the parenchyma cells, but the cause of the latter is far from apparent. It is generally assumed to be due to a lack of the tonic action of sunlight, which brings about a retardation of growth in sun plants. The evidence in favor of this view is not conclusive, and it seems probable at least that the elongation of the parenchyma cells takes place under conditions which favor the mechanical stretching of the cell wall, but inhibit the proper growth of the wall by intussusception. It is hardly necessary to state that the reduced photosynthetic activity of shade plants favors such an explanation. Whatever the cause, the advantage that results from the elongation of the internodes is apparent. Leaves interfere less with the illumination of those below them, and the leaves of the branches are carried away from the stem in such a way as to give the plant the best possible exposure for its aggregate leaf surface.