Research methods in ecology

by Frederic E. Clements

PHOTOHARMOSE

ADJUSTMENT

172. Light as a stimulus. In nature, light stimuli are determined by intensity and not by quality. A single exception is afforded by those aquatic habitats where the depth of water is great, and in consequence of which certain rays disappear by absorption more quickly than others. In forests and thickets, where the leaves transmit only the green and yellow rays, it would appear that the light which reaches the herbaceous layers is deficient in red and violet rays. The amount of light transmitted by an ordinary sun leaf is so small, however, that it has no appreciable effect upon the quality of the light beneath the facies, which is diffuse white light that has passed between the leaves. Indeed, it is only in the densest forests that distinct sunflecks do not appear. Coniferous forests, with a light value less than .005, which suffices only for mosses, lichens, and a few flowering plants, show frequent sunflecks. This is convincing evidence that the light of such habitats is normal in quality. It warrants the conclusion that in all habitats with an intensity capable of supporting vascular plants the light, no matter how diffuse, is white light. The direction of the light ray is of slight importance in the field, apart from the difference in intensity which may result from it. In habitats with diffuse light, the latter comes normally and constantly from above. Likewise, in sunny situations, direction can have little influence, since both the direction and the angle of the incident rays change continually throughout the day, and the position of the leaf itself is more or less constantly changed by the wind. The influence of duration upon the character of light stimuli is difficult to determine. There can be no question that the time during which a stimulus acts has a profound bearing upon the response that is made to it. In nature the problem is complicated by the fact that light stimuli are both continuous and periodic. The duration of sunlight is determined by the periodic return of night as well as by the irregular occurrence of clouds. Since one is a regular, and the other at least a normal happening, it is necessary to consider duration only with respect to the time of actual sunlight on sunny days, except in the case of formations belonging to regions widely different in the amount of normal sunshine, i. e., the number of cloudy days. In consequence, duration is really a question of the intensities which succeed each other during the day. The differences between these have already been shown to fall within the efficient difference for light, and for this reason the ratio between the light intensity of a meadow and of a forest is essentially the ratio between the sums of light intensity for the two habitats, i. e., the duration. The latter is of importance only where there is a daily alternation between sunshine and shadow, as at the edge of forest and thicket, in open woodland, etc. In such places duration determines the actual stimulus by virtue of the sum of preponderant intensities. The periodicity of daylight is a stimulus to the guard cells of stomata, but its relation to intensity in this connection is not clear.

The amount of change in light intensity necessary to constitute an efficient stimulus seems to depend upon the existing intensity as well as upon the plant concerned. Apparently, a certain relative decrease is more efficient for sun plants than for shade plants. At least, many species sooner or later reach a point where a difference larger than that which has been efficient no longer produces a structural response. This has been observed by E. S. Clements (l. c.) in a number of shade ecads. For example, a form of Galium boreale, which grew with difficulty in a light value of .002, showed essentially the leaf structure of the form growing in light of .03, while the form in full sunlight showed a striking difference in the leaf structure. In considering the light stimuli of habitats, it is unnecessary to discuss the stimulus of total darkness upon chlorophyllous plants, although this is of great importance in experimental evolution and in control experiment. The normal extremes of light intensity, i. e., those within which chlorenchym can function, are full sunshine represented by 1, and a diffuseness of .002, though small flowering plants have once or twice been found in an intensity of .001. The maximum light value, even on high mountains, never exceeds 1 by more than an inconsiderable amount, except for the temporary concentration due to drops of dew, rain, etc. It seems improbable that the concentrating effect of epidermal papillae can do much more than compensate for the reflection and absorption of the epidermis. Experimental study has shown that the maximum intensity in nature may be increased several, if not many times, without injurious results and without an appreciable increase in the photosynthetic response, thus indicating that the efficient difference increases toward the maximum as well as toward the minimum.

173. The reception of light stimuli. Rays of light are received by the epidermis, by which they are more or less modified. Part of the light is reflected by the outer wall or by the cuticle, particularly when these present a shining surface. Hairs diffract the light rays, and hairy coverings consequently have a profound influence in determining stimuli. The walls and contents of epidermal cells furthermore absorb some of the light, especially when the cell sap is colored. In consequence of these effects, the amount of light that reaches the chlorenchym is always less than that incident upon the leaf, and in many plants, the difference is very great. According to Haberlandt[^[16]], the epidermal cells of some shade plants show modifications designed to concentrate the light rays. Of such devices, he distinguishes two types: one in which the outer epidermal wall is arched, another in which the inner wall is deeply concave. Although there can be no question of the effect of lens-shaped epidermal cells, their occurrence does not altogether support Haberlandt’s view. Arched and papillate epidermal cells are found in sun plants where they are unnecessary for increasing illumination, to say the least. A large number of shade plants show cells of this character, but in many the outer wall is practically a plane. Shade forms of a species usually have the outer wall more arched or papillate, but this is not always true, and, in a few cases, it is the lower epidermis alone that shows this feature. Finally, a localization of this function in certain two-celled papillae, such as Haberlandt indicates for Fittonia verschaffelti, does not appear to be plausible.

The epidermis merely receives the light; the perception of the stimulus normally occurs in those cells that contain chloroplasts. The cytoplasm of the epidermal cells, as well as that of the chlorenchym cells, is sensitive to light, but the response produced by the latter is hardly discernible in the absence of plastids, except in those plants which possess streaming protoplasm. The daily opening and closing of the stomata, which is due to light, is evidently connected with the presence of chloroplasts in the guard cells. Naturally, the perception of light and the corresponding response occur in the epidermis of many shade and submerged plants which have chloroplasts in the epidermal cells. Such cases merely serve to confirm the view that the perception of light stimuli is localized in the chloroplast. In conformity with this view, the initial response to such stimuli must be sought in the chloroplast, and the explanation of all adaptations due to light must be found in the adjustment shown by the chloroplasts.

174. Response of the chloroplast. The fundamental response of a plastid to light is the manufacture of chlorophyll. In the presence of carbon dioxide and water, leucoplasts invariably make chlorophyll, and chloroplasts replace that lost by decomposition, in response to the stimulus exerted by light. The latter is normally the efficient factor, since water is always present in the living plant, and carbon dioxide absent only locally at most. Sun plants which possess a distinct cuticle, however, produce leucoplasts, not chloroplasts, in the epidermal cells, although these are as strongly illuminated as the guard cells, which contain numerous chloroplasts. This is evidently explained by the lack of carbon dioxide in the epidermis. This gas is practically unable to penetrate the compact cuticle, at least in the small quantity present in the air. The supply obtained through the stomata is first levied upon by the guard cells and then by the cells of the chlorenchym, with the result that the carbon dioxide is all used before it can reach the epidermal cells. This view is also supported by the presence of chloroplasts along the sides and lower wall of palisade cells, where there is normally a narrow air-passage, and their absence along the upper wall when this is closely pressed against the epidermis, as is usually the case. Furthermore, the leaves of some mesophytes when grown in the sun develop a cuticle and contain leucoplasts. Under glass and in the humid air of the greenhouse, the same plants develop epidermal chloroplasts but no cuticle. This is in entire harmony with the well-known fact that shade plants and submerged plants often possess chloroplasts in the epidermis. Although growing in different media, their leaves agree in the absence of a cuticle, and consequent absorption of gases through the epidermis. The size, shape, number, and position of the chloroplasts are largely determined by light, though a number of factors enter in. No accurate studies of changes in size and shape have yet been made, though casual measurements have indicated that the chloroplasts in the shade form of certain species are nearly hemispherical, while those of the sun form are plane. In the same plants, the number of chloroplasts is strikingly smaller in the shade form, but exact comparisons are yet to be made. The position and movement of chloroplasts have been the subject of repeated study, but the factors which control them are still to be conclusively indicated. Light is clearly the principal cause, although there are many cases where a marked change in the light intensity fails to call forth any readjustment of the plastids. The position of air-spaces as reservoirs of carbon dioxide and the movement of crude and elaborated materials from cell to cell frequently have much to do with this problem. Finally, it must be constantly kept in mind that the chloroplasts lie in the cytoplasm, which is in constant contact with a cell wall. Hence, any force that affects the shape of the cell will have a corresponding influence upon the position of the chloroplasts. When it is considered that in many leaves these four factors play some part in determining the arrangement of the plastids, it is not difficult to understand that anomalies frequently appear.

It may be laid down as a general principle that chloroplasts tend to place themselves at right angles to rays of diffuse light and parallel to rays of sunlight. This statement is borne out by an examination of the leaves of typical sun and shade species, or of sun and shade forms of the same species. Cells which receive diffuse light, i. e., sponge cells, normally have their rows of plastids parallel with the leaf surface, while those in full sunlight place the rows at right angles to the surface. This disposition at once suggests the generally accepted view that chloroplasts in diffuse light are placed in such a way as to receive all the light possible, while those in sunlight are so arranged as to be protected from the intense illumination. Many facts support this statement with respect to shade leaves, but the need of protection in the sun leaf is not clearly indicated. The regular occurrence of normal chloroplasts in the guard cells seems conclusive proof that full sunlight is not injurious to them. Although the upper wall of the outer row of palisade cells is usually free from chloroplasts, yet it is not at all uncommon to find it covered by them. These two conditions are often found in cells side by side, indicating that the difference is due to the presence of carbon dioxide and not to light. In certain species of monocotyledons, the arrangement of the chloroplasts is the same in both halves of the leaf, and there is no difference between the sun and shade leaves of the same species. The experimental results obtained with concentrated sunlight, though otherwise conflicting, seem to show conclusively that full sunlight does not injure the chloroplasts of sun plants, and that the position of plastids in palisade cells is not for the purpose of protection. This arrangement, which is known as apostrophe, is furthermore often found in shade forms of heliophytes. In typical shade species, and in submerged plants, the disposition of plastids on the wall parallel with the leaf surface, viz., epistrophe, is more regular, but even here there are numerous exceptions to the rule.

The absorption of the light stimulus by the green plastid results, under normal conditions, in the immediate production of carbohydrates, which in the vast majority of cases soon become visible as grains of starch. The appearance of starch in the chloroplasts of flowering plants is such a regular response to the action of light that it is regarded as the normal indication of photosynthetic activity. The mere presence of chlorophyll is not an indication of the latter, since chlorophyll sometimes persists in light too diffuse for photosynthesis. The amount of starch formed is directly connected with the light intensity, and in consequence it affords a basis for the quantitative estimation of the response to light. Two responses to light stimuli have a direct effect upon the amount of transpiration. Of the light energy absorbed by the chloroplast, only 2.5 per cent is used in photosynthesis, while 95–98 per cent is converted into heat, and brings about marked increase in transpiration. Furthermore, in normal turgid plants, the direct action of light, as is well known, opens the stomata in the morning and closes them at night.

Fig. 38. Ecads of Allionia linearis, showing position of chloroplasts. The palisade shows apostrophe, the sponge epistrophe: 1, sun leaf (chresard, 2–5%, light, 1); 2, shade leaf (chresard, 11%; light, .012); 3, shade leaf (chresard, 11%; light, .003). × 250.

175. Aeration and translocation. The movements of gases and of solutions through the tissues of the leaf are intimately connected with photosynthesis, and hence with responses to light stimuli. Aeration depends primarily upon the periodic opening of the stomata, for, while the carbon dioxide and oxygen of the air are able to pass through epidermal walls not highly cutinized, the amount obtainable in this manner is altogether inadequate, if not negligible. The development of sponge tissue or aerenchym is intimately connected with the stomata. The position and amount of aerenchym and the relative extent of sponge cells and air-spaces are in part determined by the number and position of the breathing pores. The disposition of air spaces has much to do with the arrangement of chloroplasts in both palisade and sponge tissues. Starch formation is also dependent upon the presence of air spaces, but, contrary to what would be expected, it seems to be independent of their size, since sun leaves, which assimilate much more actively than shade leaves, have the smallest air spaces. From this fact, it appears that the rapidity of aeration depends very largely upon the rapidity with which the gases are used. Translocation likewise affects the arrangement of the chloroplasts and the formation of starch. According to Haberlandt, it also plays the principal part in determining the form and arrangement of the palisade cells. Chloroplasts are regularly absent at those points of contact where the transfer of materials is made from cell to cell, though this is not invariably true. Since air passages are necessarily absent where cell walls touch, it is possible that this disposition of the plastids is likewise due to the lack of aeration. Translocation is directly connected with the appearance of starch. As long as all the sugar made by the chloroplasts is transferred, no starch appears, but when assimilation begins to exceed translocation, the increasing concentration of the sugar solution results in the production of starch grains. The latter is normally the case in all flowering plants, with the exception of those that form sugar or oil, but no starch. The constant action of translocation is practically indispensable to starch formation, since an over-accumulation of carbohydrates decreases assimilation, and finally inhibits it altogether. In consequence, translocation occurs throughout the day and night, and by this means the accumulated carbohydrates of one day are largely or entirely removed before the next.

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.

Fig. 40. Isophotophyll of Allionia linearis, showing diphotic ecads: 1, light 1; 2, light .012; 3, light .003. × 130.

Fig. 41. Isophotophyll of Helianthus pumilus, showing isophotic ecad: 1, sun leaf; 2, shade leaf (light .012). × 130.

179. Modification of the epidermis. The development of epidermal chloroplasts in diffuse light is the only change which is due to the direct effect of light. This does not often occur in the shade ecads of sun species, but chloroplasts are regularly present in the epidermis of woodland ferns and of submerged plants. The slight development of hairs in sciophilous plants is an advantage, but it must be referred to the factors that determine water loss. The significance of epidermal papillae in increasing the absorption of light by shade plants has already been discussed. The questions as to what factor has called forth these papillae and what purpose they serve must still be regarded as unsettled. The increased size of the epidermal cells, which is a fairly constant feature of shade ecads, seems to be for the purpose of increasing translocation and transpiration, and to bear no relation to light. The extreme development of the cells of the epidermis in Streptopus and Limnorchis, which grow at the edge of mountain brooks, has been plausibly explained by E. S. Clements as a contrivance to increase water loss. The presence of a waxy coating, such as that found upon the leaves of Impatiens aurea and I. pallida, is clearly to prevent the wetting of the leaf and the consequent stoppage of the stomata. In regard to the latter, different observers have noted that the number of the stomata is greater in sun than in shade leaves. This holds generally for sun and shade species, but it is most clearly indicated by different ecads of the same species. In Scutellaria brittonii, the sun form possesses 100 stomata per square millimeter, but in the shade these are reduced to 40 per square millimeter; the sun leaf of Allionia linearis has 180 stomata to the square millimeter, the shade leaf 90. In the stable leaf of Erigeron speciosus, however, the number of stomata is the same, 180 per square millimeter, for sunlight and for diffuse light. The presence of the larger number of stomata in the plant exposed to greater loss, which at first thought seems startling, is readily explained by the more intense photosynthetic activity in the sun. Since the absorption of gases is the primary function of the stomata, and transpiration merely secondary, it is evident that sun plants must have more stomata than shade plants. This is further explained by the fact that the small air passages of sun leaves necessitate frequent inlets, which are less necessary in shade leaves with their larger air spaces. In shade plants, moreover, the decrease in the number is compensated in some measure by the ability of the epidermal cells to absorb gases directly from the air.

Fig. 42. Diphotophylls of Quercus novimexicana: 1, sun leaf; 2, shade leaf of the same tree (light .06). × 130.

180. The differentiation of the chlorenchym. The division of the chlorenchym into two tissues, sponge and palisade, is the normal consequence of the unequal illumination of the leaf surfaces. Exceptions to this rule occur only in certain monocotyledons, in which the leaf tissue consists of sponge-like cells throughout, and in those stable species that retain more or less palisade in spite of their change to diffuse light. The difference in the illumination of the two surfaces is determined by the position of the leaf. Leaves that are erect or nearly so usually have both sides about equally illuminated, and they may be termed isophotic. Leaves that stand more or less at right angles to the stem receive much more light upon the upper surface than upon the lower, and may accordingly be termed diphotic. Certain dorsiventral leaves, however, absorb practically as much light on the lower side as upon the upper. This is true of sun leaves with a dense hairy covering, which screens out the greater part of the light incident upon the upper surface. It occurs also in xerophytes which grow in light-colored sands and gravels that serve to reflect the sun’s rays upon the lower surface. In deep shade, moreover, there is no essential difference in the intensity of the light received by the two surfaces, and shade leaves are often isophotic in consequence. From these examples it is evident that isophotic and diphotic leaves occur in both sun and shade, and that the intensity of the light is secondary to direction, in so far as the modification of the leaf is concerned.

The essential connection of sponge tissue with diffuse light is conclusively shown by the behavior of shade ecads, but further evidence of great value is furnished by diphotic leaves, and those with hairy coverings. The sponge tissue, which in the shade leaf is due to the diffuse light of the habitat, is produced in the hairy leaf as a consequence of the absorption and diffraction of the light by the covering. In ordinary diphotic leaves, the absorption of light in the palisade reduces the intensity to such a degree that the cells of the lower half of the leaf are in diffuse light, and are in consequence modified to form sponge tissue. The sponge tissue of the diphotic leaf is just as clearly an adaptation to diffuse light as it is in those plants where the whole chlorenchym is in the shade of other plants or of a covering of hairs. As is indicated later, all these relations permit of ready confirmation by experiment, either by changing the position of the leaf or by modifying the intensity or direction of the light.

Fig. 43. A plastic species, Mertensia polyphylla, showing the effect of water upon the sponge: 1, chresard 25%; 2, chresard 12%. × 130.

The preceding discussion makes it fairly clear that sponge tissue is developed primarily to increase the light-absorbing surface. Because of its direct connection with photosynthesis, the sponge tissue is the especial organ of aeration, also, and since it shows a high development of air spaces for this purpose, it is inevitably concerned in transpiration. It seems to be partly a coincidence, however, that the sponge is found next to the lower surface upon which the stomata are most numerous. This is indicated by artificial ecads of Ranunculus sceleratus, in which sponge tissue is unusually developed, although the stomata are much more numerous upon the upper surface. Palisade tissue is apparently developed primarily as a protection against water loss, particularly that due to the absorption of light by the chloroplast. The small size of the intercellular passages between palisade cells likewise aids in decreasing transpiration. The fact that leaves with much palisade tissue transpire twice as much as shade leaves is hardly an objection to this view, as Hesselmann (l. c., 442) would think. It is readily explained by the intense photosynthesis of sun plants, which makes necessary an increase, usually a doubling, in the number of stomata, in consequence of which the transpiration is increased.

Fig. 44. A stable species, Erigeron speciosus: 1, sun leaf; 2, shade leaf (light .03). × 130.

Fig. 45. Spongophyll of Gyrostachys stricta (light 1). × 130.

181. Types of leaves. Isophotic leaves are equally illuminated and possess more or less uniform chlorenchym. Diphotic leaves are unequally illuminated, and exhibit a differentiation into palisade and sponge tissues. They may be distinguished as isophotophylls and diphotophylls respectively.[^[18]] Isophotic leaves fall into three types based upon the intensity of the light. The staurophyll, or palisade leaf, is a sun type in which the equal illumination is due to the upright position or to the reflection from a light soil, and in which the chlorenchym consists wholly of rows of palisade cells. The diplophyll is a special form of this type in which the intense light does not penetrate to the middle of the leaf, thus resulting in a central sponge tissue, or water-storage tissue. The spongophyll, or sponge leaf, is regularly a shade type; the chlorenchym consists of sponge cells alone. For the present at least it is also necessary to refer to this group those monocotyledons which grow in the sun but contain no palisade tissue. Diphotic leaves always contain both palisade and sponge, though the ratio between them varies considerably. Diphotophylls are characteristic of sunny mesophytic habitats. They are frequent in xerophytic habitats as well as in woodlands where the light is not too diffuse. In the case of stable species, this type of structure sometimes persists in the diffuse light of coniferous forests. Floating leaves, in which the light is almost completely cut off from the lower surface, are also members of this group. Submerged leaves, on the other hand, are spongophylls.

182. Heliophytes and sciophytes. The great majority of sun plants possess diphotophylls. This type is represented by Pedicularis procera (fig. 32). Plants with isophotophylls are found chiefly in xerophytic places, though erect leaves of this type occur in most sunny habitats. The staurophyll, in which the protection is due to the extreme development of palisade tissue, is illustrated by Allionia linearis (fig. 40) and Bahia dissecta (fig. 33). The diplophyll, which is characterized by a central band of sponge tissue or storage cells, is found in Mertensia linearis (fig. 34). The form of the spongophyll that is found in certain monocotyledons is shown by Gyrostachys stricta (fig. 45). The spongophyll (fig. 38:3, 39:2) is frequent among plants of deep shade, but as the leaf sections of Allionia (figs. 38, 40) and Quercus (fig. 42) show, the diphotophyll is the rule in shade ecads.

Experimental Evolution

183. Scope. The primary task of experimental evolution is the detailed study, under measured conditions, of the origin of new forms in nature. As a department of botanical research that is as yet unformed, it has little concern with the host of hypotheses and theories which rest merely upon general observation and conjecture. A few of these constitute good working hypotheses or serve to indicate possible points of attack, but the vast majority are worthless impedimenta which should be thrown away at the start. It is the general practice to speak of evolution as founded upon a solid basis of incontestible facts, but a cursory examination of the evidence shows that it is drawn, almost without exception, from observation alone, and has in consequence suffered severely from interpretation. With the exception of De Vries’s work on mutation, sustained and accurate investigation of the evolution of plants has been lacking. As a result, botanical research has been built high upon an insecure foundation, nearly every stone of which must be carefully tested before it can be left permanently in place. In a field so vast and important as evolution, experiment should far outrun induction, and deduction should enter only when it can show the way to a working hypothesis of real merit. The great value of De Vries’s study of mutation as an example of the proper experimental study of evolution has been seriously reduced by the fact that the “mutation theory” has carried induction far beyond the warrant afforded by experiment. The investigator who plans to make a serious study by experiment of the origin of new plant forms should rest secure in the conviction that the most rapid and certain progress can be made only by the accumulation of a large number of unimpeachable facts, obtained by the most exact methods of experimental study.

The general application of field experiment to evolution will render the current methods of recognizing species quite useless. It will become imperative to establish an experimental test for forms and species, and to apply this test critically to every “new species.” Descriptive botany, as practiced at present, will fall into disuse, as scientific standards come to prevail, and in its place will appear a real science of taxonomy. In the latter the criteria upon which species are based will be obtained solely by experiment.

184. Fundamental lines of inquiry. There are two primary and sharply defined fields of research in experimental evolution, namely, adaptation in consequence of variation (and mutation), and hybridization. The latter constitutes a particular field of inquiry, which is not intimately connected with the problems of evolution in nature. In the study of specific adaptation, two questions of profound importance appear. One deals with the effects of ancestral fixity or plasticity in determining the amount of modification produced by the habitat. These are fundamental problems, and a solution of them can not be hoped for until exact and trustworthy data have been provided by numerous experimental researches. It thus becomes clear that the principal, if not the sole task of experimental evolution for years to come is the diligent prosecution of accurate and prolonged experiment in the modification of plant forms. It seems inevitable that this will be carried on along the lines that have already been indicated. Plants will be grown in habitats of measured value, or in different intensities of the same factor. The relation between stimulus and adjustment will form the basis of careful quantitative study, and the final expression of this relation in structural modifications will find an exact record in drawings, photographs, exsiccati, and biometrical measures. The making of an accurate and complete record of the whole course of each experiment of this sort is an obligation that rests upon every investigator. Studies in experimental evolution will prove time-consuming beyond all other lines of botanical research, and the work of one generation should appear in a record so perfect that it can be used without doubt or hesitation as a basis for the studies of the succeeding generation.

185. Ancestral form and structure. The significance of the fact that some species have been found to remain unaltered structurally under changes of habitats that produced striking modifications in others has already been commented upon. It is hardly necessary to indicate the important bearing which this has upon evolution. The very ability of a plant to undergo modification, and hence to give rise to new forms, depends upon the degree of fixity of the characters which it has inherited. Stable plants are less susceptible of evolution than plastic ones. The latter adapt themselves to new habitats with ease, and in each produce a new form, which may serve as the starting point of a phylum. There is at present no clue whatever as to what calls forth this essential difference in behavior. This is not surprising in view of the fact that there have been no comparative experimental studies of stable and plastic species. Until these have been made, it is impossible to do more than to formulate a working hypothesis as to the effect of stability, and an explanation of the forces which cause or control it is altogether out of the question.

186. Variation and mutation. New forms of plants are known to arise by three methods, viz., variation, mutation, adaptation. The evidence in support of these is almost wholly observational, and consequently more or less inexact, but for each there exist a few accurate experiments which are conclusive. Origin by variation and subsequent selection is the essence of the Darwinian theory of the origin of species. According to this the appearance of a new form is due to the accumulation, and selection, through a long period, of minute differences which prove advantageous to the plant in its competition with others in nature, or are desirable under cultivation. Slight variations appear indiscriminately in every species. Their cause is not known, but since they are found even in the most uniform habitats, it is impossible to find any direct connection between them and the physical factors. In the case of origin by mutation, the new form appears suddenly, with definite characteristics fully developed. Selection, in the usual sense of the term, does not enter into mutation at all, though the persistence of the new form is still to be determined by competition. Mutations are known at present for only a few species, and their actual appearance has been studied in a very few cases. Like variations, they are indiscriminate in character. The chief difference between them is apparently one of degree. Indeed, mutation lends itself readily to the hypothesis that it is simply the sudden appearance of latent variations which have accumulated within the plant. De Vries regards constancy as an essential feature of mutation, but the evidence from the mutants of Onagra is not convincing. Indeed, while there can be no question of the occurrence of mutation in plants, a fact known for many years, the facts so far brought forward in support of the “mutation theory” fall far short of proving “the lack of significance of individual variability, and the high value of mutability for the origin of species.”[^[19]] Mutations do not show any direct connection with the habitat, but their sudden appearance suggests that they may be latent or delayed responses to the ordinary stimuli. Origin by adaptation is the immediate consequence of the stimuli exerted by the physical factors of a habitat. This fact distinguishes it from origin by variation, or by mutation. The new form may appear suddenly, often in a single generation, or gradually, but in either case it is the result of adaptation that is necessarily advantageous, because it is the result of adjustment to controlling physical factors. Origin by adaptation is perhaps only a special kind of origin by variation, but this might be said with equal truth of mutation. New forms resulting from adaptation are like those produced from mutation, in that they appear suddenly as a rule and without the agency of selection. They are essentially different, inasmuch as their cause may be found at once in the habitat, and since a reversal of stimuli produces, in many cases at least, a reversion in form and structure to the ancestral type.

A valid distinction between forms or species upon the basis of constancy is impracticable at the present time. It is doubtful that such a distinction can ever be made in anything like an absolute sense, since all degrees of fluctuation may be observed between constancy and inconstancy. In all events, it is gratuitous to make constancy the essential criterion in the present state of our knowledge. So little is certainly known of it that it is equally unscientific to affirm or to deny its value, and even a tentative statement can not be ventured until a vast amount of evidence has been obtained from experiment. Accordingly, there is absolutely no warrant, other than tradition, for limiting the term species to a constant group. In the evolutionary sense, a species is the aggregate ancestral group and the new forms which have sprung from it by variation, mutation, or adaptation. It should not be regarded as an isolated unit for purposes of descriptive botany; indeed, its use in this connection is purely secondary. It is properly the unit to be used in indicating the primary relationships which are the result of evolution.

On the basis of their actual behavior in the production of new forms, species may be distinguished as variable, mutable, or adaptable. The new form which results from variation is a variant; the product of mutation is a mutant, and that of adaptation, an ecad. The following examples serve to illustrate these distinctions. Machaeranthera canescens, judging from the numerous minute intergrades between its many forms, is a variable species, i. e., one in which forms are arising by the gradual selection of small variations. It apparently comprises a large number of variants, M. canescens aspera, superba, ramosa, viscosa, etc. Onagra lamarckiana is a mutable species: it comprises many mutants, e. g., Onagra lamarckiana gigas, O. l. nanella, O. l. lata, etc. Galium boreale is an adaptable species: it possesses one distinct ecad, Galium boreale hylocolum, which is the shade form of the species.

187. Methods. The best of all experiments in evolution are those that are constantly being made in nature. Such experiments are readily discovered and studied in the case of origin by adaptation; variants present much greater difficulties, while mutants are very rare under natural conditions. The method which makes use of these experiments may be termed the method of natural experiment. The number of ecads which appear naturally in vegetation is limited, however, and it is consequently very desirable to produce them artificially, by the method of habitat culture. This method, while involving more labor than the preceding, yields results that are equally conclusive, and permits the study of practically every species. The method of control culture, which is carried on in the planthouse, naturally does not possess the fundamental value of the field methods. It is an invaluable aid to the latter, however, since it permits the physical factors to be readily modified and controlled. All these methods are based on the indispensable use of instruments for the measurement of physical factors.