In the middle of the past century the ideas of Darwin and of Wallace concerning the transformation of living organisms loosed a flood of speculation as to the underlying causes of evolution. Many of the theories propounded entirely out-ran the facts, and the natural reaction has been to lay aside hypotheses and logical deductions and look for specific evidence in every field. Biologists everywhere have quietly set themselves to the task of finding data that have any possible bearing on this major problem. This does not mean that interest in the subject has grown less but rather that the immensity of the problem is seen in a truer perspective.

Of the post-Darwinian speculation the theories of Gulick (1887) and of Romanes (1897) on physiological isolation had little specific evidence in their support. Guilck's (1905) painstaking observations on the distribution of land mollusks in the Hawaiian Islands, extended by Crampton (1916), give much evidence as to the importance of geographical isolation but have little bearing on physiological isolation. In the third volume of Darwin, and after Darwin, which deals with isolation and physiological selection, Romanes cites few observations and no experimental evidence. Very little experimental work had been done on the problem at this time, although the experiments on mixed pollinations reported by Darwin in Cross and Self-Fertilization in the Vegetable Kingdom and the numerous observations on self-sterility and enforced cross-fertilization were certainly well known to Romanes and his editor, C. L. Morgan. Gulick mentions Darwin's experiments indicating a prepotency of foreign pollen but has little to say about them. In spite of the handicap of unsupporting data, their deductions are essentially sound and stand today as the best exposition of this undoubtedly important factor in evolution.

The idea of physiological isolation, the incompatibility of different species and of groups within species, was originally developed to offset the assumed swamping effect of free intercrossing. For a long time it was believed that cross-fertilization among all the members of a species in one locality would reduce all the individuals to a dead level of sameness. Geographical isolation was one means by which this was supposed to be prevented, but this separating influence was not applicable to many clear cases of diversification in small areas. When Mendel's experiments showed clearly that the determiners of hereditary characters are not blended together in a common melting pot after intercrossing, and that individual genes exist indefinitely to be sorted out later unchanged by their association with other germinal elements, physiological isolation seemed to lose some of its importance.

At the present time there is a tendency to overlook the real contribution of Gulick and of Romanes to evolutionary theory. The facts presented in the preceding chapters, meager as they are, supply the sort of evidence they needed to make their plausible arguments convincing. Physiological incompatibility not only is associated with specific differences but in an incipient form is present at the beginning of variation. It is the starting point of a differentiation that finally leads to organisms incapable of uniting.

Both Gulick and Romanes clearly recognized all forms of discriminate isolation. They gave all possible credit to geographical isolation. Sexual selection, where physiological preference is exercised among organisms differing in form, color, or other more intangible traits, was also fully considered. In plants it was noted that intercrossing may be prevented by any slight difference in time of flowering, of topographical stations, or even by differences in the instincts and preferences of their visitors, in the case of flowers which depend on insects for their fertilization. Due probably to the lack of evidence, very little attention was given to assortative mating, especially in the lower forms, although it was mentioned

Both authors laid their principal emphasis upon natural selection and physiological isolation rather than on other forms of homogamy. They showed that natural selection is one kind of isolation, since by this process the fittest are restrained from uniting with the less fitted by the exclusion of the latter. Physiological isolation was not proposed as an alternative hypothesis to natural selection but as a supplement to it. The principal argument of these writers was that isolation due to natural selection helps to transform a species but some other kind of isolation is necessary to branch a species. According to their idea, monotypic evolution was possible with natural selection alone acting on variations of whatever origin, but not polytypic evolution.

Whatever basis the advocates of physiological isolation had for their theory, aside from deductive reasoning, was largely derived from the observations of Jordan (1873) and of Nägeli (1875). Both of these botanists noted the variations within species and saw that various groups or subspecies existed side by side in the same areas. Jordan carried his ideas farther and found that many of these subspecific groups bred true and "when they did so they were always more or less cross-infertile inter-se." I cannot find that Jordan had any positive evidence upon which to base this statement, which is quoted from Romanes (loc. cit.), other than the observation in nature that no intermediate forms were seen and hence intercrossing was assumed not to take place. Their lack of knowledge of the laws of inheritance at that time makes this observation of little value.

Romanes and his editor, C. L. Morgan (1897), completely ignored the phenomenon of self-sterility and a great deal of evidence as to the widespread occurrence of this and other means of enforcing cross-fertilization which were well known at that time. They were inclined to be impatient with Darwin for not giving more attention to physiological isolation. Probably the reason why he had not attached more importance to it was because his own experiments did not indicate that plants had any tendency toward homogamy within small groups. In fact, his results, which have been reviewed here in chapter IV, showed the directly opposite effect. Plants of the same species differing in minor characters seemed to be fertilized more readily by pollen from different types than by their own pollen.

It has been pointed out that Darwin's experiments upon this problem were neither critical nor conclusive. The results presented in the preceding chapters prove that there is a noticeable tendency toward homogamy in plants only slightly differentiated, as the theory of physiological isolation demands; but it must be credited to Darwin that he was consistent with the results as he found them. Darwin in his Animal and Plants under Domestication refers to experiments and observations of Gärtner, Naudin, and others, which indicate a restriction to cross-fertilization; but he is justified in not attaching much importance to them.

Gärtner compared the amount of seed produced when different varieties of the same species, particularly those belonging to the genus Verbascum, were self-pollinated and cross-pollinated, and found that plants fertilized with pollen from their own kind with respect to color of flowers were more fertile. The flowers were emasculated in both types of pollinations. In a large number of pollinations the crosses yielded from 10 to 30 percent fewer seeds than the interpollinations. Scott, cited by Darwin, gives similar results obtained at the Botanic Gardens in Edinburgh. He repeated the experiments of Gärtner with Verbascum; and in every case where varieties of the same species were pollinated by their own kind of pollen and that from a differently colored variety of the same species on different flowers, the dissimilar unions were less productive of seed than the unions of similar plants.

Naudin (1865) observed that various forms of Citrullus which were interfertile when artificially pollinated and were naturally readily cross-fertilized nevertheless remained unchanged when grown close together for many years. Most cucurbits are not easily kept true to type.

How difficult it is to judge correctly such results is shown by another observation of Gärtner, who for many years grew two varieties of maize in his garden, one a dwarf type with yellow seed, the other a tall sort having red seeds, and states that they did not cross spontaneously although artificial cross-pollinations set a small number of seeds. Possibly, crossing took place but was not observed, since the red color is in the pericarp and is not immediately affected by cross-pollination. This, however, does not account for the plants remaining unchanged over a period of years if seed was harvested each year and used for the following sowing. At the Connecticut Agricultural Experiment Station some of the most diverse types of maize known have been readily crossed. The giant-seeded Cuzco corn from Peru is reciprocally quite fertile with the tiny-seeded rice pop corn having ears about the size of one's thumb (Jones, 1924). To what extent they intercross naturally was not determined.

So many influences can affect the production of seed that none of these observations of the early hybridizers afford critical evidence for reduced fertility from intercrossing. Taken with the results from mixed pollinations where a selective action is positively shown, they are at least suggestive.

The importance of this tendency for similar forms to take part in fertilization more readily than dissimilar forms should not be overemphasized. In its incipient stages the discrimination is by no means rigorous. Cross-fertilization does occur even if less easily, and the greater reproductive ability of the cross-bred organisms for the first few generations due to hybrid vigor tends to nullify the tendency toward homogamy. Moreover, crossing over and recombination of the factors that make the final result of fertilization selective tend to set up an ever increasing number of different types, each of which is in some degree physiologically isolated from all others. Every possibility is thus given for natural selection to sort out those combinations of characters that are most favorable to survival; and as they become more fixed and stable, they are also more effectively isolated from related types.

Biologists are generally agreed that the principal importance of sex is to make organisms more plastic. The advantages have been such that sexual reproduction is now established as the prevailing method for the renewal of organisms in both kingdoms, at a sacrifice of economy and speed of multiplication. It is not strange, then, that many accessory devices have been developed to insure the fulfilment of the function for which so much has been expended. Self-sterility, dioecism, dichogamy, hetero-styly, and other floral devices favoring cross-fertilization—all help to bring about germinal mixing.

The reverse tendency, that of self-prepotency, has been shown to occur in several widely separated species. In maintaining these two opposing tendencies, nature is not necessarily working at cross-purposes. Bi-parental inheritance with the inclination toward exogamy serves to bring about variability. The preferential mating of similar kind operates to make germinal mixing discriminative. Based upon evidence from the corners of the phylogenetic triangle—angiosperm, protozoan, and man—homogamy is entitled to an authoritative part in evolutionary theory.

It has been argued that the qualities which separate species are essentially different from the visible variations the geneticists are now describing in terms of "genes." Bateson has voiced this position in his address in 1922 before the American Association for the Advancement of Science.

"Analysis [of the hereditary traits of animals and plants] has revealed hosts of transferable characters. Their combinations suffice to supply in abundance series of types which might pass for new species, and certainly would be so classed if they were met with in nature. Yet critically tested we find that they are not distinct species and we have no reason to suppose that any accumulations of characters of the same order would culminate in the production of distinct species. Specific differences therefore must be regarded as probably attaching to the base upon which these transferables are implanted of which we know absolutely nothing at all. Nothing that we have witnessed in the contemporary world can colorably be interpreted as providing the sort of evidence required."

Lack of knowledge concerning the primary factors of evolution undoubtedly makes the stand of the skeptic a safe one at the present time. Critical doubt is certainly more conducive to real progress than the enthusiastic speculation of the preceding decades. At the same time the evidence reviewed here has a bearing on this problem.

Bateson's criterion of specific differences is largely based upon the production of sterile offspring. The conditions laid down are such that they are very difficult of fulfilment. He says: "The production of an indubitably sterile hybrid from completely fertile parents, which have arisen under critical observation from a common origin; this is the event for which we wait." If such an individual arose in one generation in a bisexual organism, it would by definition be incapable of continued reproduction and would be lost. In hermaphroditic organisms it would ordinarily not be crossed back with the parental race and would not be discovered.

There is no evidence that complete intergroup sterility arises suddenly; nor should it be expected. Sexual incompatibility is the final criterion of germinal differentiation whether or not this is preceded by sterility of the offspring. The preliminary results obtained by Plough with Drosophila which show reduced fertility in outcrossing as compared to matings within the strain indicate a tendency in the direction of intergroup sterility. The individuals differing in chromosome number from their parents, obtained in experimental cultures by different investigators, also give an indication of specific differences. The tetraploid Datura, obtained by Blakeslee, which is almost completely sterile with its diploid parent and the Raphanus-Brassica hybrids of Karpechenko come the nearest to meeting the requirements of complete physiological isolation of a new form from its parental stock of any evidence obtained up to the present time and show the undoubted importance of chromosome rearrangements in the separation of species.

But it is evident that chromosome aberration is not the primary reason for the beginning of incompatibility. It may be one of the results, rather than the cause, of some change that leads to incompatibility. Certain it is, that intergroup sterility has arisen in many organisms in which no chromosome variation has taken place. The central problem is to find out the nature of those changes which have been the starting-point for the separation of different groups.

It has been shown conclusively in several widely separated species of plants that fertilization takes place less readily when the gametes come from unlike forms than when homogeneous unions are made. The discrimination becomes more pronounced as the germinal differences of the uniting individuals become greater. There is here exhibited the working of a tendency which acts to set individuals apart. This manifestation of physiological isolation in plants is paralleled in the assortative mating of animals from the lowest to the highest, and the physiological and morphological variations involved in this discrimination are accompanied by physiological differences that make cross-fertilization more and more difficult. When carried far enough, there is created an impassable physiological barrier separating different groups.

The differences shown by the separating groups are the usual qualitative and quantitative characters, as far as can be judged, which have been described in terms of "Mendelian units." The factors directly concerned in the differential pollen-tube growth in plants may be of an entirely different nature from those hereditary units that make the visible differences, but there is some evidence that this is not the case; and whatever these factors are they are transferable. Nothing is known as to how they originate, and very little as to how they act. They merit the closest study, since we have exhibited in these factors something that seems to be concerned with the physiological differences that separate species. Individual members of species, which were once sexually compatible, are now diverse in form and behavior and show a marked tendency toward sexual incompatibility. The fact that there is a condition of measurable physiological isolation is reason to suppose that the accumulation of characters of the same order will culminate in different groups being clearly set apart.