_Oryctolagus cuniculus_). Three methods of injection of antigen were
used in various combinations: intravenous, subcutaneous, and
intraperitoneal. Injection schedules used in the production of each
antiserum are listed in Table 1. Both formolized and "native" antigens
were used. Each rabbit received one or more series of four injections,
each injection being administered on alternate days and doubling in
amount: 0.5 ml., 1.0 ml., 2.0 ml., and 4.0 ml. In all but two
instances more than one series of injections was necessary to produce
a useful antiserum. More than two series, however, resulted in little
or no improvement of the reactivity of the antiserum.
The injection-series were separated by intervals of eight days. On the
eighth day after the last injection of each series, 10 ml. of blood
were withdrawn from the main artery of the ear of the rabbit, and the
antiserum was used in a homologous precipitin test to determine its
usefulness. If the antiserum contained sufficient amounts of
antibodies to conduct the projected tests, the rabbit was completely
exsanguinated by cardiac puncture, by using an 18-gauge needle and a
50 ml. syringe. The whole blood was placed in clean test tubes and
allowed to clot. It was allowed to stand at 2 deg.C. for 12 to 18 hours so
that most of the serum would be expressed from the clot. The serum was
then decanted, centrifuged to remove all blood cells, sterilized in a
Seitz filter, bottled in sterile vials, and stored at 2 deg.C. until used.
Methods of Serological Testing
The precipitin reaction is the most successful of the serological
techniques thus far devised for systematic comparisons. The reaction
occurs because antigenic substances introduced into the body of an
animal cause the formation of antibodies which precipitate antigens
when the two are mixed. The antisera which are produced show
quantitative specificities in their actions; therefore, when an
antiserum containing precipitins is mixed with each of several
antigens, the reaction involving the homologous antigen (that used in
the production of the antiserum) is greater than those reactions
involving the heterologous antigens (antigens other than those used in
the production of the antiserum). Furthermore, the magnitudes of the
reactions between the antiserum and the heterologous antigens vary
according to the degrees of similarity of these antigens to the
homologous one.
The method of precipitin testing follows that outlined by Leone
(1949). The Libby (1938) Photronreflectometer was used to measure the
turbidities developed by the interaction of antigen and antiserum.
With this instrument parallel rays of light are passed through the
turbid systems being measured. Light rays are reflected from the
suspended particles to the sensitive plate of a photoelectric cell;
this generates a current of electricity which causes a deflection on a
galvanometer. The deflection is proportional to the amount of
turbidity developed and readings may be taken directly from the scale
of the instrument.
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 | .. | .. |
--------------------------+----+----+----+----+----+----+----+
[Illustration: FIGS. 10-13. Graphs of precipitin reactions
illustrating 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_.]
[Illustration: FIGS. 14-17. Graphs of precipitin reactions
illustrating 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 assemblage.
FIG. 17. Serological relationships of _Carpodacus_ and _Spinus_ with
_Richmondena_ and _Junco_.]
[Illustration: FIGS. 18-21. Graphs of precipitin reactions
illustrating 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.]
[Illustration: FIG. 22. Two views of a model illustrating
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 . . . . _Passer_ Z . . . . _Zonotrichia_]
[Illustration: FIG. 23. Two additional views of the model shown in
fig. 22 illustrating 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 . . . . _Passer_ Z . . . . _Zonotrichia_]
Within the richmondenine-emberizine-thraupid assemblage, _Junco_
and _Zonotrichia_ constitute 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 assemblage 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.