Popular Science Monthly/Volume 69/September 1906/Discontinuous Variation in Pedigree-Cultures
| DISCONTINUOUS VARIATION IN PEDIGREE-CULTURES[1] |
By Dr. D. T. MACDOUGAL
DIRECTOR, DEPARTMENT OF BOTANICAL RESEARCH, CARNEGIE INSTITUTION OF WASHINGTON
HEREDITY may be defined as that appurtenant function of living matter by which qualities, characters and capacities are transmitted through successive generations. The absolute identity, or measurable expression, of the inherited characters may be qualified by partial and individual fluctuations about a norm in a continuous series in the progenies, and these characters, singly or in groups, may be subject to dominations, recessions, integrations and resolutions in hybridizations, or to various forms of combination, actual or apparent. In the case of discontinuous variations or mutation, single or unit characters, or constellations of them, may be activated, or converted into a latent or perlatent condition.
Methods of Investigation
Owing to the stimulus of recent discovery, attention is focused at the present time upon the ultimate result of fluctuating variability as influenced by various agents, in the origin and fate of hereditary strains, species, races or physiologically unified groups of organisms, and upon the probable part played in the matter by the saltatory movements, which are being brought to notice so plentifully in all quarters. In connection with the last-named feature, the behavior of the individual qualities or unit-characters in hybridizations are being studied with enormous zeal as offering a ready analysis of the action of inheritance. The comparative ease with which hybrid combinations of plants are effected, and the simplicity of the subsequent resolutions in the progeny, render this phase of the subject easy of attack, and results are being obtained, which, if one may judge from recent literature, do not seem available to many writers.
It needs but a moment's consideration to bring the realization that the entire subject offers some of the most abstruse and difficult problems in the whole realm of biological science. Intricate and elusive in their physiological complexity, we may hope to uncover the main factors by perfected methods in research upon the ultimate mechanical basis of heredity coupled with a refinement of technique in dealing with the course of inheritance as we trace it from generation to generation, eliminating guesswork as to parentage, and prophecy as to offspring. The first is steadily yielding results of undoubted importance and is bringing about a renewed interest in the functional relations of the components of the protoplast with respect to the inheritance of characters. The second method, that of statistical observations and experimental methods in pedigreed cultures, has given such notable results in the hands of various investigators, that it may be truly asserted that it may not be outclassed in value by any form of research yet used in investigations in natural history.
As an explorer, do you wish to ascertain the source, direction, character, rate of flow and confluence of a river across your route? Surely, you do not reasonably attempt it by an examination of a single reach, or from a photograph of a single waterfall. Even so surely you may not gauge the possibilities of development, or estimate the potentiality or method of action of groups of characters, embraced in a hereditary strain, guided by dimly recognized forces for thousands of years, by the measurement of a preserved specimen. Physiological problems demand analytic methods of observation of living material.
What ridicule might we not heap upon a botanist who attempted to make a study of geotropism from consideration of the dried material in a herbarium. The existence of such a form of reaction might indeed be recognized, but what futile inferences might be drawn as to its mechanism or the nature of the results. It seems unnecessary and superfluous to call attention to a generalization so obvious; yet that the necessity is not lacking is shown by the material that crowds the pages of our technical magazines and popular periodicals.
Inadequate Treatment of the Subject
Before proceeding with the main thesis it will be profitable to notice some of the most glaring of the inadequacies of treatment which have been recently exhibited, and to call attention to certain unsupported statements which so far have gone unchallenged.
A vice-president of the American Association for the Advancement of Science, in a recent address, has taken occasion to call up the mutation theory, and assumes to have given it a test by "reexamining certain groups of birds and mammals, of which I had previously made systematic studies, for the purpose of discovering evidence, if such exists, of the formation of species by mutation." This author says that "for a quarter of a century I have been an earnest field student of plants in relation to geographic environment. These studies have convinced me that with plants as with animals the usual way in which new forms (subspecies and species) are produced is by gradual progressive development of minute variations." In regard to this comprehensive statement I may say that I have read practically every thing that Dr. Merriam has put into print concerning his extensive and thorough field work in the west, and that I yield to none in my admiration of the wide inclusiveness of his results and the profitable manner in which he has treated incidentally the general features of the occurrence and distribution of plants. This appreciation is heightened by the fact that I have spent many seasons in the regions covered by him during the last fifteen years, and that I have carried on experimental work in the field and at the Desert Laboratory with many of the species which are included in his generalizations. I have not been able to come upon the evidence, or record of evidence, upon which his sweeping statements relative to plants are based, although detailed studies upon the relation of plants to environmental factors have been in progress for some time.
Dr. Merriam does not find any evidence to support the conclusion that species arise by mutation. It would be a matter of great surprise if he had. It would be as reasonable to have demanded of him the solutions of problems of respiration from his preparations and field notes. Once a mutant has appeared, no evidence of its distribution can be taken to account conclusively for its origin. Although I have had many mutants under experimental observation, I should not be able to speak with reasonable certainty as to the origin of any of them, had I not ascertained it by guarded pedigree-cultures. It also follows that the systematists who announce and describe new forms as mutants, simply from preserved specimens, or from individuals, the origin of which is not a matter of careful observation, are following a wholly unwarranted practise. Several months ago I had occasion to say "that the 'naturalists,' as some zoologists term themselves, having made the greatest number of essays to offer a universal interpretation of the problems of distribution, are to be credited with the greatest number of defenseless assumptions. In all genetic and evolutionary researches too much emphasis can not be laid upon the basal fact that the physiological and morphological natures of the two great classes of living things are so widely divergent that the derivation of universal biological principles from their apparently concurrent behavior must be made with the greatest caution." Nowhere does this find better exemplification than in the unedifying results of a recent discussion of isolation as a factor in evolutionary action. A number of zoologists have assumed to speak of the distribution of plants, with apparently no basis except 'general information' to the effect that closely related species do not have the same habitat. This has been variously put, but the general meaning is as given. Now such a conclusion is so widely inapplicable, and is so loosely guarded, as to be wholly without value as a statement of a principle in plant geography and floristics. That the actual mechanical contiguity of two forms competing for the same conditions of habitat would result in some stress is to be taken for granted, but this vegetative struggle would by no means be severe enough in any case to eliminate one from any region. If the advocates of the idea that closely related species do not occupy the same region take this ground on the assumption that hybridization disturbances would follow, here again would be an unwarranted assumption. Readiness of hybridization is by no means a measure of consanguinity, and any slight difference of habit, so small perhaps as not to be capable of description, might ensure pure fertilization. In the case of forms differing by one or a few characters, Mendelian splitting might operate to maintain the forms even if hybridization did occur.
The accompanying photograph of Opuntia fulgida and O. mammillata presents two forms so closely related that the latter has been taken as a variety of the former by some botanists, but it has been found to be a distinct and physiologically unified strain, and worthy of specific rank. These two forms are widely intermingled, though of course not many instances of actual contact such as appears in the illustration are to be found. The flora in the vicinity of the Desert Laboratory, at Tucson, presents scores of similar examples among other species representing many genera.
Aside from such misinterpretations, a prolific source of confusion lies in the widely different conceptions as to the nature of the taxonomic units used in zoological and botanical writings, as a consequence of which we have some zoologists calling attention to the supposed fact that certain botanists of differing views have no real conception as to the nature of 'species' and 'varieties.' Such statements serve the useful purpose of emphasizing the disadvantageous prejudices under which their authors labor.
While such misunderstandings contribute to hinder progress and confuse the subjects, the basal and underlying fault consists in the fact that taxonomic and geographic methods are not in themselves, or conjointly, adequate for the analysis, or solution of genetic problems. The inventor did not reach the solution of the problem of construction of a typesetting machine by studying the structure of printed pages, but by actual experimentation with mechanisms, using printed pages only as a record of his success. Likewise no amount of consideration of fossils, herbarium specimens, dried skins, skulls or fish in alcohol may give any actual proof as to the mechanism and action of heredity in transmitting qualities and characters from generation to generation, although from such historical data the general trend or direction of succession may be traced.
Misinterpretation of Results
Beyond such mistaken attempts at an analysis of the problems, there is another series of difficulties that interferes materially with the advancement of knowledge of the subject. This consists in careless, prejudiced or mistaken interpretation of results, having the force in some instances of actual misrepresentation. Such demonstrations do no final harm, yet they befog a difficult subject: with 'opinions' and 'beliefs,' they are quite out of place in any scientific discussion.
Since I have had Œnothera Lamarckiana, one of the plants which offer a favorable example of discontinuous variation in unit-characters, under cultivation for several years, I am disposed to regard Dr. D. S. Jordan's recent statement concerning this plant as of the above character. He says "it is not at all unlikely that the original Œnothera Lamarckiana found in the field near Amsterdam was a hybrid stock, a product of the florist, the behavior of its progeny being not unlike that which appears in the progeny of hybrids. It is moreover known that the seeds of hybrids of an American species, probably Œnothera biennis, the common evening primrose, with other American species produced by Mr. Burbank, have been in the past years sold in the cities of Germany." The well-informed botanist will be in doubt as to whether these statements are supposed to lie in 'the plane of ether' or are to be taken literally. If seriously meant, carelessness as to the existence of records of the plant in question in several localities, longprevious to the beginning of the activity of any living horticulturist, is shown. Moreover, material from these localities has been found to be in a mutative condition. It is unnecessary to cite facts so readily accessible in any well-appointed botanical library. The following will be of interest in connection with the statements quoted above:
1. Numerous and repeated hybridizations between O. biennis and other species have been made without obtaining anything resembling O. Lamarckiana in anatomy or behavior. Several unit-characters are exhibited by O. Lamarckiana not found in any other species.
2. O. grandiflora obtained pure in its original habitat is now giving off mutants in the cultures in the New York Botanical Garden, after p manner generally similar to O. Lamarckiana.
3. The close examination of the evening-primroses shows that the several species are extremely localized. O. grandiflora, discovered by John Bartram in 1778, was unknown except in gardens, until rediscovered in the original habitat by Professor S. M. Tracy in 1904. O. parviflora has been known in Europe at least since 1759 and has not been seen in America until 1905, when some undoubted indigenous specimens were found in Maine. During the interim the reasoning shown above would have designated these two species as 'hybrid' or 'garden products.'
It is true, of course, that similar finality of evidence with regard to O. Lamarckiana has not yet been obtained, but it does not seem 'unlikely' that it may come to hand when we are able to organize a search for it as well-directed as for the species named. Collectors are few in the region to be covered, and it might be many years before it could be traced to its habitat, even if it occupied a large area. To those interested in suppositions, however, the following from Miller ('Figures of the Most Useful and Uncommon Plants described in the Gardener's Dictionary exhibited on Three Hundred Copper Plates Accurately Engraven after Drawings taken from Nature,' Vol. II., 1760) will suggest the need of caution in the matter. He says regarding the "Tree Primrose with oval spear-shaped indented Leaves, and Flowers proceeding from the Wings of the Leaves on the Upper Part of the Stalk":
This plant is also a Native of North America; but "was the first species of the Genus which was brought to Europe, so is more commonly seen in the Gardens than any of the other species. In some Parts of Europe, this is spread about from the Gardens in such Plenty, that it might be supposed a Native there. In a small Wood near Haerlem in Holland this Plant covered the Ground insomuch that many skilful Persons supposed it was a Native of that Place. But it may be easily accounted for; because the Gardeners who live near that Place, are chiefly Florists, and they annually change the Earth of the Beds in their Gardens; so by carrying out of their old Earth from their Beds, in which many of the Seeds were scattered the Plants came up there; and those being suffered to scatter their Seeds, had filled the whole Wood with the Plants.
This differs from the first Sort, (described and figured as O. biennis) in having broader Leaves; the Stalks grow taller, and the Flowers are much larger. Both these Sorts will thrive in the Smoak of London better than most Plants.The appended descriptions and the plate (No. 189, dated 1757) very fittingly characterize O. biennis and O. Lamarckiana, and as the descriptions were made before O. grandiflora, the only known species which might be confused with O. Lamarckiana, was discovered, this evidence is serious enough to give one pause in ascribing a hybrid origin to the last-named. Meanwhile the mutation-theory, based on the conception of unit-characters, will neither stand nor fall by suppositions or proof as to the ancestry of O. Lamarckiana. If this plant and all of its derivatives were obliterated from our records, the facts in our possession well warrant current conclusions as to unit-characters and their appearance and disappearance in hereditary strains in saltatory fashion.
A certain literary freedom of expression is well illustrated by the following citation from a recent article by Dr. Jordan in this magazine, in which he says: The last sentence offers a fair example of the misrepresentation to which Mr. Burbank's horticultural work has been so profusely the object. A similar progeny of a hybrid oak is included in experimental cultures in New York, and the observer may readily see that the physiological possibilities are not exhausted in either case. To illustrate the possible variations in form would require many millions of individuals, as may be seen when a simple computation shows that seven single differentiations would require more than sixteen thousand individuals for their exemplification, if the characters behaved as indivisible units. If, however, qualities or characters are capable of modification or variation, as indicated by the quotation, the number of different forms of any organ of the entire plant would be so large as to make estimates useless. Then again by what extended experimentation have the conceivable variations in every function been ascertained?
A wider range of literary license prevails in some recent articles by Mr. E. A. Ortmann. Among other inaccuracies he says:
Although somewhat familiar with 'Die Mutationstheorie' and 'Species and Varieties,' no explanation occurs to me to account for this mistaken statement. A few combination forms were found and faithfully recorded by de Tries, but these were certainly not intergrades, whatever might be said of them.
Mr. Ortmann's discussions introduce a novel feature, in his estimate of the futility of experimental methods, which has the sole merit of boldness, coming at a time when the greater number of workers in the subject are turning from discussions and statements of opinion to actual observations. A mistrust is shown by him of experiments 'under artificial and unnatural conditions, as for instance in the botanical garden, or with domesticated forms.' Several months ago the following characterization of this attitude was given in a paper on the subject:[2]
So far nothing has been offered which would tend to disprove these conclusions.
It is by no means intended to maintain that the stream of heredity may not be altered by the action of external agents, and the possibility of having such changes ensue in experimental cultures beckons with alluring finger to the observer. So important do I hold this aspect of the matter, that a series of experiments, yet in progress, were begun previous to the mutation cultures, and these tests have been continued and expanded until one plant is now undergoing culture in New York, Jamaica, and in connection with the Desert Laboratory, at sea-level and altitudes of 2,300. 5.000. 6,000 and 8,000 feet, and under conditions widely different from those prevalent in its original habitat. If at the end of the decade, this, or any other of the species under test, shows any transmission of the characters induced by the various localities, the care and work necessary in the experiments will be richly rewarded. In this comment reference is had to factors presented by tillage, or entering into the environment of plants in their native habitats.
Announcement has been previously made that mutations, breaks, saltations or discontinuous action may be caused in inheritance by forces external to the protoplasts and cells, which are the true bearers of the hereditary characters (see p. 17).
The technique of the methods by which such changes are induced might be simulated by the action of gaseous emanations, from the soil. radio-action, foreign pollen, or by the stings and incisions of insects, but certainly these possible factors would lie as potent with wild as with cultivated plants, as may be seen in the description of the manner in which such changes have been produced.
The Method of Pedigree-cultures
The importance of pedigree-cultures of plants as a means of tracing the course and action of heredity has been notably emphasized by recent investigations, and it will be profitable to go into a detailed statement of the manner in which experiments of this kind are carried out. One of the most striking developments of methods of research in botany has been the continually increasing extent to which taxonomists are having recourse to observations upon growing material in botanical gardens and experimental grounds. In the determination of difficult problems of relationship, it is becoming more and more customary to secure the individuals representing the doubtful forms and cultivate them under identical conditions, thus securing data for comparison and analysis, representing all stages of development of the sporophyte from the seedling to the mature fruit. The record of important questions which have been solved in this manner is a long one, and includes the investigations of Alexis Jordan on Draba, Sargent on Cratægus, Wittrock on violets. Britton and Rose on Crassulaceæ and scores of other less extensive researches.
If the observer becomes interested in the hereditary action of his plants in addition to a comparison of their anatomical and physiological qualities, it then becomes necessary to follow his plants from generation to generation to ascertain to what extent and in what manner variation may ensue.
The first step in this work is to secure purely fertilized seeds. Hybridization is not common among plants except in a few genera, yet in tests which must continue for a number of years every precaution must be used to ensure accuracy of results. The observer, therefore, covers the unopened flower buds of the individuals from which he wishes to procure seeds with bags of paper, or other suitable tissue, and then makes sure that pollination is secured spontaneously, or by band, with no danger of admixture of any kind.
In due time the ripened seeds, with photographs, notes and proper herbarium material, are taken from the parental individuals. With the first lot of seeds on hand, the next step is to make a pure culture from them. To do this a quantity of soil of the proper consistency is secured, and while in a moist condition is heated to the boiling point of water in an oven on two succeeding days, or, better still, to a higher temperature in an autoclav for four or five hours. The treated soil may now be stored to be used as wanted, but at all times it must be guarded from possible contamination by the introduction of foreign seeds.
Seed-pans, of earthenware, or shallow wooden boxes are next secured and thoroughly washed in clean water and filled with sterilized soil, after which they are set in place in a cold frame, or in a greenhouse or germination chamber. As each pan is to be used it is taken to a special operating table, and the selected seeds are sown directly from the packet, so that from three to five hundred (in the case of small seeds) are evenly distributed over the surface. A thin even layer of earth is sifted over the seeds, a wooden label is affixed to the pan, giving all necessary data, and the pan is returned to its place in the culture room. If more than one species is being tested at the same time, the greatest care must be used to prevent admixture, and the remote separation of the pans may lie necessary. The splashing of 
water from a hose, the contact of the nozzle or of the spout of a sprinkler, the careless brushing of the sleeve or the hand against the dirt, may result in the transference of seeds and the vitiation of the results of years of labor and care, especially if a complicated series of tests is under way.
Having observed all the above precautions, the seeds finally sprout in due time, unfold the seed-leaves and begin development. The remote possibility is to be taken into account that the parental individuals may have been hybrids and that this, the second, generation may illustrate the resolutions, combinations, dominancy and recessivity of the ancestral characters. If, however, no such differentiation be encountered the observer has before him a progeny which by the multiplication of his seed-pans may be made to include as many individuals as might be found in a great geographical area during any season. The accurate examination of this material, and of that offered by succeeding generations may reveal evidence of the highest interest, bearing upon various problems in heredity. It is to be noted that if guarded seeds were not obtained for the beginning of the test, the more important work must await the second generation under culture for its beginning.
In even the first examination of the progeny of any physiologically unified strain it will be evident that some diversity of form and appearance is present. This variability is generally of the fluctuating type, that is, the entire number of individuals present may be arranged in a series with respect to any quality. Thus, if the one with the narrowest leaf-blades and the one with the widest are placed at opposite poles, the others may be arranged in a continuous series which shades by the smallest increments from one to the other. In making such an arrangement, it will be found that the greater number resemble, or lie near, an individual or group of individuals at some place along the line between the two extremes. Statistical measurements of this or any other quality may be made, and the position of the norm of the type determined accurately, as well as the range of variability. In like manner, the correlation between any two characters or qualities may be worked out. Now, having in hand such a mass of data from the initial culture, a fair basis is had for taking up various questions. Thus a succeeding generation might be grown in soil deficient of any nutritive factor, or poor in all of them, in rich soil, or in a substratum highly charged with any element, or at unusual temperatures for the purpose of determining the extent and manner in which the range of variability may be altered by such special treatment, and this may be accentuated by the continuance of the test in successive progenies.
Then again the pedigree-culture offers a fair opportunity for a demonstration of the influence of the effects of selection upon the range of variability and the mean value of any quality. Comparative cultures of seeds taken from the widest-leaved individuals with those of the narrower leaves will tend to show the result of such selection, especially if the selection is continued through several generations. In all these tests it is assumed that the seeds for the following generations should in all instances be guarded as directed above and sown in sterilized soil. With such close series of cultures the question of self-and close-fertilization might arise, and here again the culture affords invaluably exact material for a test of a subject upon which but little definite information exists.
The possibilities of the pedigree-culture are by no means exhausted with covering the above points, especially if the plants chosen for the objects happen to be capable of vegetative reproduction, that is by cuttings or slips, in which case comparisons should be made of progenies grown from seeds of the plant with wide leaves with individuals grown, from slips of the same, and also compared with others from the extreme end of the series.
It is by undiscriminating discussions of horticultural operations involving seed-selection and hybridization, followed by vegetative propagation, that the public mind has become confused as to the nature and
possibilities of selection, notably by the 'popular' and pseudoscientific descriptions of the thorough work of Mr. Burbank. By successive selections certain features, such as size or quality, of a fruit are forced to a maximum development perhaps in a single plant, or may be in hundreds, after which the desirable quality is carried along, or propagated, in quantity by cuttings of the original plant, thus excluding a possibility of a return to the average habit of the species.
Or, in hybrid combinations and resolutions, the desirable constitution of some horticultural form may be secured only after the most highly complicated and repeated crossing, with a result too complex to be easily analyzed. With one desirable individual at hand which produces nuts, berries, cherries, apples, potatoes or plums, or timber, it may be made to produce hundreds and thousands exactly like it merely by using its buds and branches for grafting and budding or propagating. The use of seeds of the desirable form, either from the original or from any of its derivatives, might give play to all the complex activity of the splitting of hybrids and of the free play of fluctuating variability. During the course of study of the fluctuating variability of a species, by means of successive generations grown from purely fertilized seeds, the observer may be so fortunate as to encounter individuals which do not fit into his series by reason of the possession of some quality not visible in the greater number of the progeny, or by the lack of such qualities. Thus in a progeny of red-flowered plants one may he encountered which has white bloom, or an individual with laciniate leaves may come in a pure progeny the remainder of which has entire leaves,
or a wholly glabrous specimen may be in a hairy progeny, or an individual may depart from the progeny in all these particulars. In either case it is apparent that the variability here is not one of the modification of a character, but by the total accession or loss of a character, and the variability is therefore a discontinuous one. In the progenies in which such variants have been seen hitherto, they form a proportion never larger than six or eight per cent, of the whole number and generally in much less quantity. If such mutants or saltatory variants be found, they should be closely followed, as they may furnish the observer with facts of the greatest value. Care should be taken to secure purely fertilized seeds and a minute anatomical examination should be made of the entire plant, with special respect to the characters in which it appears to differ from the mass of the fluctuating variants around the type.
The seeds secured from such mutants should be sown as directed above, and as many individuals as possible secured for comparison with the parental type, which should be continued as before. If now the individuals of the mutant 
Fig. 5. Rosette of Raimannia odorata. progeny are placed in a series with respect to any given quality, statistical observations may show whether it is included within the range of fluctuating variability of the parental type. The question therefore as to whether a plant is a continuous or discontinuous variant is one of simple measurement and estimation of qualities, not a matter of opinion. With that simple question easily disposed of, the investigator may next turn his attention to the correlations, a phase of the question of vivid interest, since the sparse data at hand seem to point to a wider range of variation and less degree of correlation in such mutants than in the parental type. That is to say, instead of mutants being derived from forms showing widely fluctuating variability, we have them appearing in the progenies of species in which the range of variation is small and the correlations close, while the derivatives themselves swing through a wide range of fluctuations. 
Fig. 6. Rosetta of Mutant of Raimannia odorata induced by Chemical Treatment of Ovules.
As a complementary means of investigation of the constancy and independence of the various characters dealt with in selection and mutation as observed above, hybridization forms an invaluable means of analysis, and is to be constantly resorted to in all stages of the various phases of the work, since in the observation of the behavior of unit-characters in combination and after resolution the clearest appreciation of their character may be reached.
Still another phase of variability is that in which the greater portion of an individual will be found to comply with the requirements of a continuous series, but which bears a branch or portion of a branch which differs notably by the loss or acquisition of characters. Although all discontinuous variations or mutations are essentially vegetative, yet these are generally termed so exclusively. It is to be noted that such sports, or mutants, when closely fertilized, come true to their aberrant characters. Among other numerous conclusions sustaining this point, one which has recently come to notice again is that of Kerner,[3] who said:
In the context the author is careful to point out the limitations of such a method of origin of forms.
Actual pedigrees from such sports or aberrant branches have been tested, in one instance by de Vries and in two others by myself, with the unanimous result that they were found to be constant to their aberrant characters.
Having carried on such pedigree-cultures with a large number of species for several years and having encountered some which did and others which did not give rise to aberrant individuals, attention was directed to the possibility of inducing changes in the hereditary elements in such a manner that the qualities transmitted would be altered or destroyed. A theoretical consideration of the subject seemed to indicate that the changes constituting the essential operation of mutation ensued in a stage previous to the reduction divisions in the embryo sac, or the pollen mother cells. It was planned therefore to subject these structures to the action of chemical agents, not ordinarily encountered by the elements in question, at a time before fertilization occurred. The tests were planned to include the use of a solution of high osmotic value, and mineral compounds, some of which are toxic in concentrated solutions and stimulating in the proportions used. The probability of success would be heightened with the number of ovules contained in any ovary operated upon, and therefore the common evening-primrose, Œnothera biennis, Raimannia odorata a relative of it 
Fig. 8. Mutant of the Common Evening Primrose, induced by Treatment of Ovules with Solutions of Zinc Sulphate. and a member of the same family, Begonia, Cleome, Abutilon, Sphæralcea and Mentzelia and others were experimented upon. Without recourse to the detail of the work, it may be stated that the use of radium preparations, sugar solutions (10 per cent.), and solutions of calcium nitrate one part in one to two thousand of distilled water with capsules of Raimannia odorata, and zinc sulphate in a stronger solution used with Œnothera biennis, was followed by very striking results. In the first-named plant, there appeared in the progeny obtained from a few capsules of one individual, several individuals which were seen to differ notably from the type with the appearance of the cotyledons, and, as development proceeded, it was evident that a mutant had appeared following the injections and nowhere else, and thus to have some direct relation to the operation. The characters of the newly arisen form were so strikingly aberrant as to need no skill in their detection. The parent was villous-hairy, the mutant entirely and absolutely glabrous, the leaves of the parent have an excessive linear growth of the marginal portions of the leaf-blades and hence become fluted; the excess of growth in the mutant lies along the midrib and the margins become revolute. The leaves are widely different in width, those of the mutant being much narrower. The parental type is of a marked biennial habit and near the close of the season the internodes formed are extremely short, which has the result of forming a dense rosette; the mutant forms no rosette by reason of the fact that the stem does not cease, or diminish its rate of elongation, and hence presents an elongated leafy stem, which continues to enlarge as if perennial. The first generation of the derivative came to bloom at the beginning of the present year, and bare mention of the existence of the derivative was given in a lecture before the Barnard Botanical Club at that time. The real value of the changes induced however lay in the transmissibility of the newly exhibited qualities. The flowers of the mutant were closely guarded and as soon as seeds were obtained these were planted to obtain a second generation. A few plants were obtained, which in every particular conformed to the new type and exhibited no return to the parental type.
Injections of the ovaries of Œenothera biennis were followed by the production of one individual, which was recognizably different from the parental type in many qualities, some of which were plainly apparent even in the earliest leaves of the seedlings. These differences have become accentuated with the adult plant and are graphically illustrated by figures 7 and 8. The succeeding generations of this mutant are yet to be tested. The parental form has been under observation for five years in cultures and in a wild condition. An aberrant form, which appears to be ever-sporting, has been previously figured, and while this form appeared in the injected or treated seeds in a normal proportion, yet the newest aberrant has not been seen elsewhere. The probability must be taken into account that it may be a mutant of rare occurrence, the cycle of which came within the experiments, but in either case it is plainly a mutant, and it only remains to be seen whether or not it was induced by the action of the zinc solution. The presumption seems to favor such an affirmative conclusion.
In finding our way about in the voluminous literature of evolution it must lie borne in mind that the subject embraces the origin and development of the universe, and that it has engaged the serious attention of workers in all branches of knowledge. The multiplicity of viewpoints has resulted in the greatest diversity of conclusions as a necessary concomitant of widely differing methods of approach to the subject. Much that has been written concerning the subject is of a purely literary or polemical character, embodying prejudices, general opinions and beliefs, putting forward conclusions drawn at long range from attempted interpretations of the results of investigations not properly considered, and brought out for the sole purpose of swaying opinion or influencing sentiment. All work of this character as well as narrow and insecurely founded investigations are futile and ineffectual except to befog the subject and hinder progress.
The problems included in a study of organic evolution are essentially physiological, and the elucidation of the mechanism and action of heredity by which qualities, characters and capacities are transmitted from generation to generation may lie accomplished only by accurate observations and experimental tests with active or living material. The examination of preserved material not in hereditary series, or the wide generalizations derived from geographic studies, may not contribute to the progress of exact knowledge of genetics, or methods and manner of inheritance.
The combined and organized efforts of all the botanists in the world concentrated upon all the herbaria in existence would add but little to existing conclusions upon this subject, if we may judge by past achievements or immediate promise, while the most precise information upon geographical distribution can be of interest only in deciphering what has been accomplished, what forms exist and where, the factors influencing their movements, and where these have probably originated.
To appreciate the mechanism of heredity an exact knowledge of the nature and behavior of the bodies which form its physical basis must be gained. To ascertain the action of heredity, statistical and accurate observations must be made upon long series of pedigreed progenies, and these must be carried out in such manner that environmental conditions may be either controlled or their influence measured. Pedigree-culture, first extensively applied by de Vries to clovers, teasels, poppies, snap-dragon and evening-primroses with such marked success, and notf used by many workers with animals as well as plants, has proved to be one of the most efficient forms of research yet used by the biologist, and its usefulness is hardly beginning to be realized. The various phases of selection, the accurate measurement of fluctuating variability, correlations, the amount and character of the influence of environmental factors, the effects of close and cross breeding, and the detection of saltatory variations may be accomplished under circumstances which allow a thorough and exact appraisement of the general physiological value of such phenomena by the use of cultures in continuous series.
While the phases of evolution are generally estimated in terms of origin or formation of species, the basal problems of heredity are not especially concerned with the taxonomic estimation given by this author or that author as to the taxonomic standing of any form, nor does it matter whether it is a subspecies, elementary species or 'real' species. The questions of evolution are to be answered by the acquisition of more accurate knowledge concerning the accession, modification or loss of functions, capacities and characters of physiologically unified groups of organisms, regardless of the necessarily more or less artificial appraisements of taxonomy. The briefest review of recent literature will reveal the widest diversity of opinion between botanists and zoologists, and also unexplainable differences among botanists and zoologists as to the species-conception. The value of discussions into which such possible differences may enter is not enhanced by this fact.
The carrying out of pedigree-cultures in New York has revealed the occurrence of discontinuous variants or mutants in Œnothera biennis, and O. grandiflora among the species tested, in addition to furnishing ample exemplification of the derivatives of O. Lamarckiana, as described by de Vries. Mutations in other genera await further test and observation, a matter which may occupy some time, before final announcement.
Despite general assertions to the contrary, no evidence has yet been obtained to prove that the influence of tillage, 'cultivation,' or the mere pressure of environmental factors has produced any permanent changes in hereditary characters of unified strains of plants.
The above is not meant as a sweeping assertion that inherited characters may not be affected by agents external to the protoplasts that bear them. On the contrary, the experiments now well advanced and conclusively verified, first announced in December, 1905[4], and here described for the first time, show that saltatory inheritance has been induced by the action of external agents upon the ovules of two species of seed-plants.[5] The alterations in question consist in the total suppression of some qualities of the parental form and the substitution therefor of new characters or of a total gain of new qualities in some instances, and the differentiating points between the parental form and the derivative are both anatomical and physiological.
The atypic form which has been tested to the second generation in one species is found to constitute a mutant in the sense in which that term is used by de Vries, and is a real and actual departure from the course of the hereditary strain. The capacity of the mutants induced in this manner for survival would depend entirely upon the environment into which they might be thrown.
If we seek a similar possible intervention of external forces which might act upon the plant unaided by man, we might find such influence coming from radio-active substances, such as spring-and rain-water, or from the effects of sulphurous and other gases which are being set free in numberless localities, or the protoplasts most nearly in contact with the egg-apparatus may well excrete substances which would produce the same effect, without regard to the forces which originally caused the disturbances in the extra-ovular tissue. Lastly it is to be said that the actual technique of injection might be imitated in a measure by the action of foreign pollen which might find lodgment on the stigmatic surfaces, and sending down tubes through the style introduce unusual substances to the vicinity of the egg-cell without participating in normal fertilization, which would ensue in the customary manner. Lastly it is to be said that it would appear that a most prolific source of such disturbances might be expected to result from the stings and lacerations of insects, or the action of parasitic fungi, both sources of the most profound morphogenic alterations in somatic tissues, profusely exemplified by the well-known gall formations of plants.
- ↑ Lecture given at Marine Biological Laboratory, Woods Holl, July 20, 1906.
- ↑ 'Heredity and Origin of Species,' Monist, January. 1906.
- ↑ 'Die Abhängigkeit der Pflanzengestalt von Klima und Boden,' Innsbruck, 1869.
- ↑ MacDougal, 'Heredity and Origin of Species.' Reprinted in advance from the Monist for January, 1906. and distributed December 18, 1905.
- ↑ The possibility is not excluded that the reagents may have affected the elongating pollen-tubes.