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 h.o.m.ologous 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 h.o.m.ologous 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 2C, 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 a.s.sume, 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 a.s.sume, 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 ill.u.s.trated 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 ill.u.s.trated further by a test (not ill.u.s.trated) involving _Zonotrichia querula_ (the h.o.m.ologous 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 h.o.m.ogeneity of pa.s.seriform birds is emphasized by the fact that the value of every heterologous reaction was more than 50 per cent of the value of the h.o.m.ologous 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 Pa.s.seriformes), the relatively high value of the heterologous reaction emphasizes the close serological correspondence of pa.s.serine 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 "h.o.m.ologizing" 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 a.n.a.lyzing 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 a.n.a.lysis 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 ill.u.s.trate 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 _Pa.s.ser_ 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 | --------------------------+----+----+----+----+----+----+----+ _Pa.s.ser domesticus_ | .. | 74 | 73 | .. | 72 | .. | .. | --------------------------+----+----+----+----+----+----+----+ _Molothrus ater_ | .. | 54 | .. | 65 | .. | 69 | 75 | --------------------------+----+----+----+----+----+----+----+ _Piranga rubra_ | .. | 77 | .. | 91 | 73 | 74 | .. | --------------------------+----+----+----+----+----+----+----+ _Carpodacus purpureus_ | 70 | 71 | .. | 61 | 93 | .. | .. | --------------------------+----+----+----+----+----+----+----+

[Ill.u.s.tration: FIGS. 10-13. Graphs of precipitin reactions ill.u.s.trating effects of formalin on antigenicity and reactivity of the extracts. For further information, see text, pp. 190-193.

FIG. 10. Reactions of unformolized antigens of _Richmondena_, _Zonotrichia_, and _Molothrus_ with anti-_Richmondena_ serum.

FIG. 11. Reactions of formolized antigens of _Richmondena_, _Zonotrichia_, and _Molothrus_ with anti-_Richmondena_ serum.

FIG. 12. Reactions of anti-_Richmondena_ serum prepared against native antigen with antigens of _Richmondena_, _Zonotrichia_, _Carpodacus_, and _Spinus_.

FIG. 13. Reactions of anti-_Richmondena_ serum prepared against formolized antigen with antigens of _Richmondena_, _Zonotrichia_, _Poephila_, _Spinus_, and _Myiarchus_.]

[Ill.u.s.tration: FIGS. 14-17. Graphs of precipitin reactions ill.u.s.trating serological relationships. For further explanation, see text, pp. 190-193.

FIG. 14. Serological relationships of _Richmondena_, _Spiza_, and _Molothrus_.

FIG. 15. Serological relationships of _Richmondena_, _Spiza_, and _Molothrus_.

FIG. 16. Serological relationships of _Carpodacus_ with the richmondenine-emberizine-thraupid a.s.semblage.

FIG. 17. Serological relationships of _Carpodacus_ and _Spinus_ with _Richmondena_ and _Junco_.]

[Ill.u.s.tration: FIGS. 18-21. Graphs of precipitin reactions ill.u.s.trating serological relationships. For further explanation, see text, pp. 190-193.

FIG. 18. Serological relationships of _Spinus_ and _Poephila_ with the richmondenines.

FIG. 19. Serological relationships of _Carpodacus_ and _Spinus_ with _Richmondena_ and _Piranga_.

FIG. 20. Serological relationships of _Poephila_ and Richmondena with the carduelines.

FIG. 21. Serological relationships of _Richmondena_ and _Spinus_ with the estrildines.]

[Ill.u.s.tration: FIG. 22. Two views of a model ill.u.s.trating serological relationships among fringillid and related birds.

For further explanation, see text, pp. 193-194.

Genera Pi ... . _Piranga_ C ... . _Carpodacus_ Po ... . _Poephila_ E ... . _Estrilda_ R ... . _Richmondena_ J ... . _Junco_ Sn ... . _Spinus_ M ... . _Molothrus_ Sz ... . _Spiza_ Pa ... . _Pa.s.ser_ Z ... . _Zonotrichia_]

[Ill.u.s.tration: FIG. 23. Two additional views of the model shown in fig. 22 ill.u.s.trating serological relationships among fringillid and related birds. For further explanation, see text, pp. 193-194.

Genera Pi ... . _Piranga_ C ... . _Carpodacus_ Po ... . _Poephila_ E ... . _Estrilda_ R ... . _Richmondena_ J ... . _Junco_ Sn ... . _Spinus_ M ... . _Molothrus_ Sz ... . _Spiza_ Pa ... . _Pa.s.ser_ Z ... . _Zonotrichia_]

Within the richmondenine-emberizine-thraupid a.s.semblage, _Junco_ and _Zonotrichia_ const.i.tute a sub-group apart from the others.

_Piranga_ and _Richmondena_ show close serological correspondence.

The present taxonomic position of _Spiza_ in the Richmondeninae, which has been questioned by Beecher (1951a:431; 1953:309), is corroborated at least insofar as the serological evidence is concerned. Certainly, serological correspondence of _Spiza_ with the richmondenine-emberizine-thraupid a.s.semblage is greater than with any other group of birds tested.

It is obvious that the serological affinities of the carduelines do not lie with the richmondenines, emberizines, or thraupids. The carduelines show greater serological correspondence with the estrildines than they do with any of the other groups tested. Further serological investigation involving other species, however, is necessary before the nearest relatives of the carduelines can be determined with certainty.

The two estrildines tested (_Estrilda_ and _Poephila_) show close serological relationship. Their nearest relatives, serologically, seem to be the carduelines. The cla.s.sification (Wetmore, 1951) that places _Pa.s.ser_ in the same family with the estrildines is not upheld by the serological data available. _Pa.s.ser_ is not, serologically, closely related to any of the birds tested. It is of interest that Beecher (1953:303-305), on the basis of jaw musculature, places _Pa.s.ser_ and the estrildines in separate families (Ploceidae and Estrildidae, respectively).

_Molothrus_ shows greater serological correspondence to the richmondenine-emberizine-thraupid a.s.semblage than to any of the other birds tested. It is definitely set apart from this group, however, and its position, serologically, is compatible with that based on evidence from other sources.

There seems to be but little argument among ornithologists that icterids, fringillids, and ploceids const.i.tute families which are distinct from one another. If, then, the serological differences between _Molothrus_ (Icteridae) and _Richmondena_ (Fringillidae), between _Molothrus_ and _Zonotrichia_ (Fringillidae), and between _Richmondena_ and _Poephila_ (Ploceidae) are indicative of family differences, there are four families represented by the birds involved. _Molothrus_ represents one family; _Piranga_, _Richmondena_, _Spiza_, _Junco_, and _Zonotrichia_, a second; _Estrilda_, _Poephila_, _Carpodacus_, and _Spinus_, a third; and _Pa.s.ser_, a fourth.

CONCLUSIONS

The heterogeneity of the Family Fringillidae has been emphasized by many authors. The relationships of the species now included in this Family have been the subject of much discussion and const.i.tute an important problem in avian systematics.