Introgressive Hybridization (1949)
THE METHOD OF EXTRAPOLATED CORRELATES
Edgar Anderson

The methods described above have been used in the field, in the experimental plot, and in actual plant breeding with a great variety of hybrid material. At first in a very tentative way, and later with increasing confidence, they have been employed to determine the putative parentage of hybrid swarms. The general method, which is here formally designated for the first time as the Method of Extrapolated Correlates, has a sound theoretical basis (Anderson, 1939b; see particularly p. 692, where the theory's application to criteria of hybridity was specifically pointed out). It was presented pragmatically by Anderson and Turrill in 1938, its application to a particular example being illustrated step by step.

The method of extrapolated correlates is based on the demonstration (set forth in detail in Chapter 3) that in a species cross all the multiple-factor characters are linked with each other (Anderson, 1939b). When well-differentiated entities hybridize, we may expect their cohesive forces to continue to operate for many successive generations in hybrid swarms. Certainly for scores, and perhaps for hundreds, of generations, we may expect to find the characters that went into the cross together still tending to stay together. By a precise and detailed examination of such populations we can discover the cohesive centers of variation still existing within them. By comparative, quantitative methods we can draw up descriptions of the original entities that must have operated to produce these centers of variation. It is possible, working with a single variable population of a species previously unknown to the investigator, to draw up a precise description of the other species which is introgressing into that population. The subsequent discovery that such a species does actually exist and could have operated in that area cannot be dismissed as a remarkable coincidence; when the prediction has been verified for a complicated series of technical details, it then becomes proof. It is even possible by this method to work with a hybrid swarm and draw up detailed descriptions of both parents when neither of them are known to the observer. Crude examples of such a prediction are given in Anderson and Turrill (1938) and in Anderson and Hornback (1946). The method has since been considerably refined. It will be illustrated below from the data presented in Riley's paper on introgression in Iris (Riley, 1938).

A portion of the data from Tables 1, 2, 3, and 4 of Riley's paper were presented (page 3) in Table 1 in a slightly simplified form. The figures for sepal lengths have been rounded off to the nearest centimeter. In Riley's paper the method of attack was to examine the two species first, and from a study of them attempt to analyze what was taking place in the hybrids. Using the method of extrapolated correlates, we shall demonstrate from these same data how one may work backwards from the introgressants, to the species from which they were derived. For the purposes of the illustration, therefore, let us suppose that only Iris hexagona var. giganti-caerulea is known to us and that we have come upon Colony H-2, which is much like that species on the whole yet is more variable and shows several variants outside the ordinary range of that species. In the discussion below, following the convention established in Chapter 1, we shall use HGC to designate Iris hexagona var. giganti-caerulea and Fulva to represent Iris fulva.

For the analysis, what we need is some simple method of determining for the whole population what characters are tending to stay together and in what patterns. We shall work with pictorialized scatter diagrams, choosing for the horizontal and vertical scales two characters each of which can be measured fairly exactly in a series of grades. In Riley's data these conditions are met by petal length and by color of sepal blade. The latter, thanks to the particular chart used by Riley, was scored in a series arranged with increasing redness from violet blue through blue violet, violet, and red violet to red. Diagramming increasing redness on the vertical axis and petal length on the horizontal axis, we produce the dots of Figs. 20 and 21 for a population of HGC and for our problem population H-2. From an inspection of these dots it is apparent that redness and petal size are tending to stick together, particularly in those individuals at the left of Fig. 21 which are outside the range of ordinary HGC. We accordingly examine Riley's table to see what other characters are varying and to see how these two extreme individuals fit into this other variation. There are five such characters, each one of which Riley scored in three grades. We add these to our large dots (each one of which represents an individual plant) by using much smaller bars at five different positions around their circumferences. Tube color is represented directly above, petal shape horizontally to the right, stamen exsertion directly below, style appendages horizontally to the left, and the presence of a crest diagonally to the left. Each of these characters can be represented with no bars for one extreme grade, with a short bar for an intermediate development, and with a, long bar for the other extreme.

FIG. 20. Pictorialized diagram of 23 plants of Iris hexagona var. giganti-caerulea, scored by the symbols shown in Fig. 23 from H. P. Riley's published data.

On the hypothesis that, if redness and small petal size came into this population from the same source, other characters may have come in with them, we assume that the peculiarities which we find tending to stay together in the two individuals at the upper left of the diagram are doing so because their genes were introduced into the population together. Since all seven of these characters are apparently multiple-factor characters, the chances are inconceivably small that the genes for all could vary simultaneously. That redness, smallness, yellow tube color, petal shape, stamen exsertion, a small style appendage, and absence of a crest all are tending to stay together in this population is most readily explained as due to the influx of whole chromosomes or of chromosome segments from a species in which these characters were tied up together.

FIG. 21. Pictorialized diagram of 23 plants from a hybrid colony studied by Riley (see Plate 1). Diagrammed from his data according to the symbols of Fig. 23. The upper-left-hand star-shaped dot represents the hypothetical species responsible for the introgression, as determined by the "method of extrapolated correlates." Further discussion in the text.

From hybrid population H-2 there are indications that these characters are so correlated. By diagramming similarly the other hybrid population H-1 (Fig. 22) in the same way we can demonstrate that these correlations hold for it and are even more strongly apparent there.

Having demonstrated the repeated existence of these complex correlations, we now proceed on the hypothesis that they are the result of introgression from a species in which all these characters were united. We can, therefore, extrapolate our data on the correlates in the hybrid population and produce a conception of what species would have been required to create such an effect. Population H-2 was very similar to HGC on the whole, and even H-1 bore a strong resemblance to it. Therefore, we need to imagine what kind of iris when crossed with HGC would yield such variants. If it produced reddish blue descendants in its cross with WC, then it must have been redder still. If it produced small flowers in combination with HGC, then it must itself have had very small flowers. In this way we may extrapolate character by character from HGC to the hybrid to the other putative species. It would have had to have been an iris with very narrow, red petals, strongly exserted stamens, a yellow tube, no crest, and small stylar appendages.

FIG. 22. Pictorialized diagram of Hybrid Colony H-1 of Plate 1, plotted from Riley's data, using the symbols of Fig. 23.

Such a species having been predicted, if we can find exactly such a one in this same area, its very existence will constitute strong evidence for the suspected hybridization. Our hypothetical introgressant, of course, proved to be Fulva. The diagram of its population plotted from Riley's data (Fig. 23) agrees exactly with our extrapolations. A series of such predictions successfully made forms almost indisputable evidence for the validity of the method of extrapolated correlates and confirms the hypothesis of introgression.

FIG. 23. Within lower-right-hand box are the symbols used in all the pictorialized scatter diagrams of Figs. 20 to 23. Upper left: 23 plants of Iris fulva, plotted from Riley's data. Note the exact correspondence with the predictions of Fig. 21.

The case of extrapolation will vary with the number of easily measured differences separating the species under observation. In a genus like Fraxinus, in which species are separated for the most part by vague and inconstant differences in texture, pubescence, etc., extrapolation will be difficult, though not impossible. The more closely related the entities involved and the more similar they are morphologically, the more difficult will it be to find differences that lend themselves to precise description and measurement. In the higher plants, however, with persistence, it has always proved possible to find suitable characters. It must be admitted that the techniques of putting such differences as leaf shape, leaf texture, and branching patterns into measurable form are still in the exploratory stage, but several that have been worked out for particular cases seem to be rather generally applicable. How far these methods can be used with other kinds of organisms it would be difficult to say. Because of the relatively simple nature of their development, plants exhibit their species differences in less complicated ways than does, for example, an insect wing or a vertebrate tooth.

In trying out such a method as that described above, one elementary fact is of great importance. If possible the work should be done in the field, at least in a preliminary way. By taking squared paper to the field it will often be possible to measure at least a few of the more obvious differences in a population and make a preliminary determination of what characters are tending to cohere in that population. As the cohering center is apprehended more and more closely, the sets of characters that go together will be more and more clearly seen. One will thus be able to collect those specimens and to concentrate on the study of those characters that are the most effective.

In interpreting and measuring the results of interspecific introgression, one of the most difficult and challenging problems is the effect of a few genes from one species when introduced into the genetic background of the other. The greater the morphological hiatus between the two hybridizing entities, the more difficult does it become to predict the impact of such a recombination or to interpret it after it has been observed. One can comparatively easily estimate the probable outcome of crossing one inbred line of maize with another and then backcrossing one or two times to the original line. It takes more experience to suggest what might be the result of such an operation upon well-differentiated species. When totally different genera (such as Zea and Tripsacum) may be concerned, the possible effect of introgression of either into the other is a research problem of no mean dimensions. One may have studied genetics for a lifetime and still be totally unable to answer the question "What would be the result of any one or two genes from Drosophila if they were introduced into Zea Mays?"

In introgression, what often seems at first sight to be the appearance of something totally new usually proves to be a recombination that one had not had the wit to anticipate. Hybridization ordinarily results not in the new, but in the unexpected. For example, brilliant-colored stems and leaves often appear when Tradescantia canaliculata suffers introgression from Tradescantia subaspera var. pilosa. Neither of these species has conspicuous plant color. Careful examination, however, shows that T. subaspera has a dull purple pigment in the epidermis—so dull that it gives the leaf and stem a general appearance of very dark green. T. canaliculata has very little color in the epidermis, but what there is has none of the dark purplish cast that characterizes T. subaspera. Introgression, therefore, brings some of the basic genes for colored epidermis into T. canaliculata, and when they operate there in the absence of the dark purple modifiers they produce a brilliant effect superficially quite different from anything in either species.

In the studies of introgression between these species it was not until after the artificial backcrosss had been made that we began to suspect the origin of the subaspera introgressants in T. canaticulata. These two species are strikingly different: T. canaliculata has a few long nodes, the uppermost of which are usually the longest. T. pilosa has many short nodes, and node length decreases progressively upwards. The introgressants of subaspera tend to have brilliant stems and leaves and a much higher node number than ordinary canaliculata. Though their nodes are somewhat shorter than in the latter, the extra number more than compensates, and the introgressants are frequently twice as tall as their unmongrelized sisters. These tallish, bright-stemmed canaliculata's superficially do not look at all like T. subaspera pilosa. It is only when careful studies are made of leaf shape, inflorescence characters, and pubescence that one finds that the whole complex in a greatly diluted form is tending to stay together in these peculiar variants.

After a few examples of introgression have been studied it is much easier to recognize introgression in other genera and in other families. With active introgression, the segregation of whole chromosomes and of chromosome segments produces an overall effect on the variability of the population which, though difficult to describe, is almost unmistakable to those who have learned what it signifies. In such a population several different characters will be varying and recombining to a degree so far beyond what happens without introgression that it is of another order of magnitude. Those who have pioneered in the analysis of introgression are sometimes accused of "seeing hybrids under every bush." The truth of the matter is that, in certain groups of plants and animals, the results of hybridization are more widespread than had previously been suspected by most biologists and that the morphological effects of hybridization upon population variability are of a peculiar sort. With a little practice these peculiarities can often be recognized, even in families of plants and in floras with which the investigator is unfamiliar. By methods like those outlined above, it is possible to apply a series of critical tests to such a varying population and make valid estimates of introgression.