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°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.