Part 8

The reaction-cells of the photronreflectometer are designed to operate with a volume of 2 ml.; therefore, this volume was used in all testing. In every series of tests the amount of antiserum was held constant and the amount of antigen was varied. The volume for each antigen dilution was always 1.7 ml., and to this was added 0.3 ml. of antiserum to make up a volume of 2 ml.

TABLE 2.--Percentage values obtained from analyses of precipitin reactions. Numerals represent relative amounts of reaction between antigens and antisera. Homologous reactions are arbitrarily valued as 100 per cent, and heterologous reactions are expressed accordingly. Comparisons are meaningful only if made within each horizontal row of values.

Table headings: Col A: Estrilda amandava Col B: Poephila guttata Col C: Piranga rubra Col D: Richmondena cardinalis Col E: Spiza americana Col F: Spinus tristis Col G: Junco hyemalis Col H: Zonotrichia querula

========================+============================================== | ANTISERA ANTIGENS +-----+-----+-----+-----+-----+-----+-----+---- | A | B | C | D | E | F | G | H ------------------------+-----+-----+-----+-----+-----+-----+-----+---- Passer domesticus | 75 | 74 | 73 | 66 | 81 | 72 | ... | 81 ------------------------+-----+-----+-----+-----+-----+-----+-----+---- Estrilda amandava | 100 | 88 | 75 | ... | 79 | 72 | 53 | ... ------------------------+-----+-----+-----+-----+-----+-----+-----+---- Poephila guttata | 95 | 100 | 77 | 67 | 87 | 81 | ... | ... ------------------------+-----+-----+-----+-----+-----+-----+-----+---- Molothrus ater | 66 | 54 | 69 | 65 | 86 | 75 | 69 | 75 ------------------------+-----+-----+-----+-----+-----+-----+-----+---- Piranga rubra | ... | ... | 100 | ... | ... | ... | ... | 89 ------------------------+-----+-----+-----+-----+-----+-----+-----+---- Richmondena cardinalis| 75 | 80 | 91 | 100 | 98 | 65 | 88 | 91 ------------------------+-----+-----+-----+-----+-----+-----+-----+---- Spiza americana | 65 | 68 | ... | 71 | 100 | 64 | 67 | 80 ------------------------+-----+-----+-----+-----+-----+-----+-----+---- Carpodacus purpureus | 70 | 71 | 71 | 61 | 89 | 93 | 53 | 70 ------------------------+-----+-----+-----+-----+-----+-----+-----+---- Spinus tristis | 72 | 74 | 73 | 60 | 89 | 100 | 60 | ... ------------------------+-----+-----+-----+-----+-----+-----+-----+---- Junco hyemalis | 64 | 56 | 74 | 65 | 87 | 68 | 100 | ... ------------------------+-----+-----+-----+-----+-----+-----+-----+---- Zonotrichia querula | 65 | 71 | ... | 67 | 89 | 75 | ... | 100 ------------------------+-----+-----+-----+-----+-----+-----+-----+----

Antigens were diluted with 0.9 per cent phosphate-buffered saline solution. Tests were run in standard Kolmer test-tube racks, each test consisting of 12 tubes. Each dilution was made on the basis of the known protein concentration of the antigen. The first tube contained an initial dilution of 1 part protein in 250 parts saline and each successive tube contained a protein dilution one-half the concentration of the preceding tube, ranging up to 1:512,000. Saline controls, antiserum controls, and antigen controls were maintained with each test to determine the turbidities inherent in these solutions. These control-turbidities were deducted from the total turbidity developed in each reaction-tube, the resultant turbidity then being considered as that which was caused by the interaction of antigens and antibodies. The turbidities were allowed to develop over a 24-hour period. In the early stages of this investigation the reactions were allowed to take place at 2 deg.C. in order to inhibit bacterial growth.

Later tests were carried out at room temperatures, and bacterial growth was prevented by the addition to each tube of 'Merthiolate' in a final dilution of 1:10,000.

Experimental Data

Corrected values for the turbidities obtained were plotted with the turbidity values on the ordinate and the antigen dilutions on the abscissa. The homologous reaction was the standard of reference for all other test reactions with the same antiserum. By summing the plotted turbidity readings, numerical values are obtained which are indices serving to characterize the curves. Such values were converted to percentage values, that of the homologous reaction being considered 100 per cent. These values, plus the curves, provide the data by means of which the proteins of the birds may be compared. Plots representative of the precipitin curves are presented in Figs. 10 to 21. For convenience each plot represents only several of the 10 curves obtained with each antiserum.

A summary of the serological relationships of the birds involved in the precipitin tests is presented in Table 2, in which percentage values are presented. Since the techniques involved in testing were greatly improved as the investigation proceeded, the summary is based solely on those tests run in the later stages of the investigation. For reasons which will become apparent in later discussion, it should be emphasized that in Table 2 comparisons may be made only within each horizontal row of values.

Discussion of the Serological Investigations

One of the problems met early in this investigation was instability of the proteins in the extracts that were prepared. Extracts in which no attempt was made to inactivate the enzymes present proved unsatisfactory. It was necessary to maintain the temperature of the "native" antigens at 2 deg.C, and all work with such antigens had to be performed at this temperature. This arrangement was inconvenient; furthermore, inactivation of the enzymes was not complete even at this low temperature, and some denaturation of the proteins took place as evidenced by the gradual appearance of insoluble precipitates in the stored vials.

The preservatives, 'Merthiolate' and formalin, were used in an attempt to inhibit the autolytic action of the enzymes present. Formalin, when added to make a final dilution of 0.4 per cent, proved to be the more satisfactory of the two preservatives and was used throughout most of the work. Formalin caused slight denaturation of some of the proteins, but this effect was complete within a few hours, after which any denatured material was removed by filtration or centrifugation. The proteins remaining in solution were stable over the period necessary to complete the investigations.

The addition of formalin reduces the reactivity of the extracts when they are tested with antisera prepared against "native" antigens and causes changes in the nature of the precipitin curves. This effect has been pointed out by Horsfall (1934) and by Leone (1953) in their work on the effects of formaldehyde on serum proteins. Their data indicate, however, that even though changes in the immunological characteristics of proteins are brought about by formolization, the proteins retain enough of their specific chemical characteristics to allow consistent differentiation of species by immunological methods. In the tests which I performed, the relative positions of the precipitin curves, whether native or formolized extracts were involved, remained unchanged (Figs. 10, 11). All data used in interpretation of the serological relationships were obtained from tests in which formolized antigens of equivalent age were used.

Only three antisera were produced against formolized antigens, all others being produced against "native" extracts. The formolized antigens seemed to have a greater antigenicity, in most instances, than did those which were unformolized, and precipitin reactions involving antisera produced against formolized antigens developed higher turbidities. The antisera produced against formolized antigens were equal to but no better than those prepared against "native" extracts in separating the birds tested (Figs. 12, 13).

The rabbit is a variable to be considered in serological tests. Two rabbits exposed to the same antigen, under the same conditions, may produce antisera which differ greatly in their capacities to distinguish different antigens. It is logical to assume, therefore, that two rabbits exposed to different antigens may produce antisera which also differ in this respect. This explains the unequal values of reciprocal tests shown in Table 2. Thus, in the test involving the antiserum to the extracts of Richmondena, a value of 71 per cent was obtained for Spiza antigen, whereas in the test involving anti-Spiza serum, a value of 98 per cent was obtained for Richmondena antigen. In Table 2, therefore, comparisons may be made only among values for the proteins of birds tested with the same antiserum.

Since the amount of any one antiserum is limited, there is, of necessity, a limit as to the number of birds used in a series of serological tests. Therefore, although the results reveal the actual serological relationships of the individual species, interpretation of the relationships of the taxonomic groups must be undertaken with the realization that such an interpretation is based on tests involving relatively few species of each group. It is reasonable to assume, however, that a species which has been placed in a group on the basis of resemblances other than serological resemblance would show greater serological correspondence to other members of that group than it would to members of other groups. Specifically, in the Fringillidae and their allies, there seems to be little reason to doubt that genera, and even subfamilies, are natural groups. This is illustrated in tests involving closely related genera: Richmondena and Spiza (Figs. 14, 15, 18), Estrilda and Poephila (Fig. 21), Spinus and Carpodacus (Figs. 12, 17, 19, 20). In each of these tests the pairs of genera mentioned show greater serological correspondence to each other than they do to other kinds involved. This point is illustrated further by a test (not illustrated) involving Zonotrichia querula (the homologous antigen) and Zonotrichia albicollis. Although this test was one of an earlier series in which difficulties were encountered (the data, therefore, were not used), it is of interest that the two species were almost indistinguishable serologically.

The serological homogeneity of passeriform birds is emphasized by the fact that the value of every heterologous reaction was more than 50 per cent of the value of the homologous reaction, except in the test involving the anti-Richmondena serum and Myiarchus (Fig. 13) in which the value of the heterologous reaction was 45 per cent. Because most ornithologists consider these genera to be only distantly related (they are in different suborders within the Order Passeriformes), the relatively high value of the heterologous reaction emphasizes the close serological correspondence of passerine birds and indicates that small consistent serological differences among these birds are actually significant. The possibility that some of the serological correspondence is due to the "homologizing" effect of formalin on proteins should not be excluded. I think, however, that this effect is not entirely responsible for the close correspondence observed here.

An additional point to consider in interpretation of the serological tests is that the techniques used tend to separate sharply species that are closely related whereas species that are distantly related are not so easily separated. In other words, comparative serological studies with the photronreflectometer tend to minimize the differences between distant relatives and to exaggerate the differences between close relatives.

In analyzing the serological relationships of the species used in this study, it becomes obvious that two or more series of tests must be considered before the birds can be placed in relation to each other. For example, the data presented in Fig. 14 indicate that Spiza and Molothrus show approximately the same degree of serological correspondence to Richmondena. This does not imply necessarily that Spiza and Molothrus are closely related. If Fig. 15 is examined, it can be determined that Richmondena shows much greater serological correspondence to Spiza than does Molothrus. Thus, an analysis of both figures serves to clarify the true serological relationships of the three genera. By reference to other series of tests involving these three birds a more exact determination of their relationships may be obtained.

To illustrate this point by a hypothetical example, two species might seem equidistant, serologically, from a third species. Additional testing should indicate if the first two species are equidistant in the same direction (therefore, by implication, close relatives) or in opposite directions (therefore, distant relatives). A single test supplies only two dimensions of a three dimensional arrangement.

It is impossible to interpret and to picture the serological data satisfactorily in two dimensions; therefore, a three-dimensional model (Figs. 22, 23) was constructed to summarize the serological relationships of the birds involved. Each of the eleven kinds used consistently throughout the investigation is represented in the model. By use of the percentage values (Table 2), each bird was located in relation to the other birds. Where possible, averages of reciprocal tests (Table 3) were used in determining distances between the elements of the model. In this way seven of the birds were accurately located in relation to each other. Lacking reciprocal tests, the positions of the other birds were determined by the values of single tests (Table 4). Although these birds were placed with less certainty, at least four points of reference were used in locating each species. At least one serological test is represented by each connecting bar in the model. The lengths of the bars connecting any two elements were determined as follows: a percentage value (Table 3 and Table 4) representing the degree of serological correspondence between two birds was subtracted from 100 per cent; the remainder was multiplied by a factor of five to increase the size of the model and the product was expressed in millimeters; a bar of proper length connects the two elements involved.

From the model it is observed that, Molothrus and Passer excluded, the birds fall into two distinct groups: one includes Piranga, Richmondena, Spiza, Junco, and Zonotrichia; the other includes Estrilda, Poephila, Carpodacus, and Spinus.

TABLE 3.--Reciprocal Values Used to Determine Distances Between Elements of the Model; Each Value Represents the Average of Serological Tests Between the Species Involved

Table Headings: Col A: Estrilda amandava Col B: Poephila guttata Col C: Richmondena cardinalis Col D: Spiza americana Col E: Spinus tristis Col F: Junco hyemalis Col G: Zonotrichia querula

==========================+====+====+====+====+====+====+====+ | A | B | C | D | E | F | G | --------------------------+----+----+----+----+----+----+----+ Estrilda amandava | .. | 92 | .. | 72 | 72 | 59 | .. | --------------------------+----+----+----+----+----+----+----+ Poephila guttata | 92 | .. | 74 | 78 | 78 | .. | .. | --------------------------+----+----+----+----+----+----+----+ Richmondena cardinalis | .. | 74 | .. | 85 | 63 | 77 | 79 | --------------------------+----+----+----+----+----+----+----+ Spiza americana | 72 | 78 | 85 | .. | 77 | 77 | 85 | --------------------------+----+----+----+----+----+----+----+ Spinus tristis | 72 | 78 | 63 | 77 | .. | .. | .. | --------------------------+----+----+----+----+----+----+----+ Junco hyemalis | .. | .. | 77 | 77 | .. | .. | .. | --------------------------+----+----+----+----+----+----+----+ Zonotrichia querula | .. | .. | 79 | 85 | .. | .. | .. | --------------------------+----+----+----+----+----+----+----+

TABLE 4.--Single Values Used to Determine Distances Between Elements of the Model; Each Value Represents a Single Test Between the Species Involved

Table headings: Col A: Estrilda amandava Col B: Poephila guttata Col C: Piranga rubra Col D: Richmondena cardinalis Col E: Spinus tristis Col F: Junco hyemalis Col G: Zonotrichia querula

==========================+====+====+====+====+====+====+====+ | A | B | C | D | E | F | G | --------------------------+----+----+----+----+----+----+----+ Passer domesticus | .. | 74 | 73 | .. | 72 | .. | .. | --------------------------+----+----+----+----+----+----+----+ Molothrus ater | .. | 54 | .. | 65 | .. | 69 | 75 | --------------------------+----+----+----+----+----+----+----+ Piranga rubra | .. | 77 | .. | 91 | 73 | 74 | .. | --------------------------+----+----+----+----+----+----+----+ Carpodacus purpureus | 70 | 71 | .. | 61 | 93 | .. | .. | --------------------------+----+----+----+----+----+----+----+