Thus, the divided or undivided conditions of the _m. obturator externus_ and the _pars interna_ of the _m. gastrocnemius_ seem to be correlated with the degrees of strength of certain movements of the leg. It is conceivable that these differences in structure are correlated with the manner in which food is obtained, the birds having the bipart.i.te muscles being those which spend the most time on the ground searching and scratching for seeds and other sorts of food.

Yet, in _Leucosticte_, a cardueline, and in _Calcarius_, an emberizine, whose foraging habits are rather similar, the structure is unlike. _Leucosticte_ does resemble the emberizines and also _Piranga_ and _Spzia_ in the extension of a band of muscle fibers from the _pars interna_ of the _m. gastrocnemius_ around the front of the knee. A band of muscle fibers of this sort strengthens the knee joint and gives still more strength to the _pars interna_. This condition has been reported in a number of birds by Hudson (1937) and is, in all probability, an adaptation for greater strength of certain leg movements. The development of this band in _Leucosticte_ seems to parallel that in the other birds studied and does not indicate relationship, since in _Leucosticte_ this band arises from the undivided muscle which (as stated above) resembles only the posterior portion of the bipart.i.te muscle described for the other birds. In the latter, the muscular band arises from the anterior part of the muscle.

Minor differences in muscle pattern, like those already mentioned, are consistent also between subfamilies, but correlation of these minor differences with function is difficult. There is the implication, however, that in all the groups except the carduelines and ploceids, the emphasis is on greater strength and mobility of the leg. In the carduelines that were studied the origin of the _m. sartorius_ does not extend so far craniad as in the other species. In the latter, at least half of the origin is from the last one or two free dorsal vertebrae; in the carduelines no more than one third of the origin is anterior to the ilium. It is conceivable that the more craniad the origin, the stronger the forward movement of the thigh would be.

In _Pa.s.ser_, _Estrilda_ and _Poephila_, and in all the cardueline finches examined, the bellies of the _m. flexor perforans et perforatus digiti II_ and the _m. flexor perforans et perforatus digiti III_ are more intimately connected than they are in the other species studied. Thus, the amount of independent action of these muscles in _Pa.s.ser_, in the estrildines, and in the carduelines probably is reduced.

In _Pa.s.ser_, the estrildines, and the carduelines the edges of the sheathlike tendon of insertion of the _m. perforatus digiti III_ are thickened; as a result the insertion appears superficially to be double but closer examination reveals that there is a fascia stretched between the thickened edges. In the other species examined, the insertion is sheathlike throughout and there are no thick areas. I cannot explain this on the basis of function. The difference, however, is obvious and constant.

Aside from the differences noted above, there were variations of muscle pattern that seem to be significant only in _Vireo olivaceus_.

In this species the central, aponeurotic portion of the _m.

iliotibialis_ is absent. The origin of the _m. adductor longus et brevis_ is from the dorsal edge of the ischiopubic fenestra and not from the membrane covering this fenestra. The origin of the _pars posticus_ of this muscle, furthermore, is fleshy and not tendinous as it is in the other species. The _m. flexor perforatus digiti II_ is larger and more deeply situated in _Vireo_ and has, furthermore, no connection with the _m. flexor hallucis longus_. The latter muscle is smaller and weaker than in any of the other species and has only one (the posterior) head of origin. The _m. flexor hallucis brevis_, on the contrary, is larger than in the other birds, compensating, probably, for the small _m. flexor hallucis longus_. In those differences, however, which separate the carduelines and ploceids from the other birds studied, _Vireo_ resembles, in every instance, the richmondenines, emberizines, tanagers, warblers, and blackbirds.

On the basis of differences in leg-musculature the species which are now included in the Family Fringillidae may be separated into two groups. One group includes the richmondenines and the emberizines; the other, the carduelines. The muscle patterns of the legs of the birds of the first group are indistinguishable from those of _Seiurus_, _Icterus_, _Molothrus_, and _Piranga_, and except for the differences noted are similar to those in _Vireo_. The carduelines, on the other hand, are similar in every point of leg-musculature to the ploceids which were studied. Thus, the heterogeneity of the Family Fringillidae, as now recognized, is emphasized by differences in the muscle patterns of the leg.

COMPARATIVE SEROLOGY

General Statement

The application of serological techniques to the problems of animal relationships has been attempted with varying degrees of success over a period of approximately fifty years. Few of the earlier studies were of a quant.i.tative nature, but within the past decade, satisfactory quant.i.tative serological techniques have been developed whereby taxonomic relationships may be estimated. The usefulness of comparative serology in taxonomy has been demonstrated in investigations of many groups wherein results obtained have, in most instances, been compatible with the results obtained by more conventional methods, such as comparative morphology. As Boyden (1942:141) stated, "comparative serology ... is no simple guide to animal relationship." However, the objectiveness of its methods, the fact that it has its basis in the comparisons of biochemical systems which seem to be relatively slow to change in response to external environmental influences, and the fact that the results are of quant.i.tative nature favor, where possible, the inclusion of data from comparative serology along with that from more conventional sources when an attempt is made to determine the relationships of groups of animals.

The application of serological methods in ornithology has not been extensive. Irwin and Cole (1936) and c.u.mley and Irwin (1941, 1944) used two species of doves and their hybrids and demonstrated that a distinction between the red cells of these birds could be made by use of immunological methods involving the agglutinin reaction. McGibbon (1945) was able to distinguish the red cells of interspecific hybrids in ducks by similar methods. Irwin (1953) used similar techniques in his study of the evolutionary patterns of some antigenic substances of the blood cells of birds of the Family Columbidae. Sasaki (1928) demonstrated the usefulness of the precipitin technique in distinguishing species of ducks and their hybrids. This technique was used successfully also by DeFalco (1942) and by Martin and Leone (1952). Working with groups of known relationships, these investigators showed that the "accepted" systematic positions of certain birds were confirmed by serological procedures. The precipitin reaction, however, has never been applied to actual problems in avian taxonomy prior to the present study.

Preparation of Antigens

Although most previous work in comparative serology in which precipitin tests were used has involved the use of whole sera as antigens, Martin and Leone (1952) indicated that tissue extracts are satisfactory as antigens and that serological differentiation can be obtained with these extracts and the antisera to them. I decided, therefore, to use such extracts in these investigations, since the small sizes of the birds to be tested made it impracticable to obtain enough whole sera.

Most of the birds used were obtained by shooting, but a few were trapped and the exotic species were purchased alive from a pet dealer.

When a bird was killed, the entire digestive tract was carefully removed to prevent the escape of digestive enzymes into the tissues and to prevent putrefaction by action of intestinal bacteria. As soon as possible (and within three hours in every instance) the bird was skinned, the head, wings, and legs were removed, and the body was frozen. Each specimen, consisting of trunk, heart, lungs, and kidneys, was wrapped separately and carefully in aluminum foil to prevent dehydration of the tissues. The specimens were kept frozen until the time when the extracts were made.

When an extract was to be prepared, the specimen was allowed to thaw but not to become warm. In the cold room with the temperature of all equipment and reagents at 2C., the specimen was placed in a Waring blender with 0.9 per cent aqueous solution of NaCl buffered with M/150 K_{2}HPO_{4} and M/150 Na_{2}HPO_{4} to a pH of 7.0. The amount of reagent used was 75 ml. of saline for each gram of tissue to be extracted. The tissues were minced in the blender, allowed to stand at 2C. for 72 hours, and the tissue residues removed by centrifugation in a refrigerated centrifuge. Formalin was added to a portion of the supernatant in the amount necessary to make the final dilution 0.4 per cent. This formolization was found to be necessary to inhibit the action of autolytic enzymes over the period of time required to complete the investigations. The effects of formolization on the antigenicity and reactivity of proteins are discussed later. It was necessary to sterilize and clarify the "native" (unformolized) extracts; this was done by filtration through a Seitz filter. These "native" substances were used only in the early stages of the investigation (see below). The filtrate was bottled and stored at 2C.

In the early stages of this investigation clarification of the formolized extract was accomplished by the same sort of filtration. It was determined, however, that centrifugation in a refrigerated centrifuge at high speeds (17,000g) served the same purpose and was quicker. The formolized extracts were bottled and also stored at 2C.

(although refrigerated storage of the formolized extracts does not seem necessary). For each extract the amount of protein present was determined colorimetrically by the method of Greenberg (1929) with a Leitz Photrometer.

Species for which extracts were prepared and the protein values of the extracts are listed in Table 1. Extracts of some species were used throughout most of the experiment; extracts of others were used only when needed for purposes of comparison.

TABLE 1.--Species from Which Extracts Were Prepared and Injection Schedules for Extracts Against Which Antisera Were Produced

==========================+==========+================================= | Protein, | SPECIES | gms. per | Injection schedules for | 100 ml. | production of antisera --------------------------+----------+--------------------------------- _Myiarchus crinitus_ | 0.65 | Series 1: Intravenous, 0.5, 1.0, (Linnaeus) | | 2.0, and 4.0 ml.

--------------------------+----------+--------------------------------- _Pa.s.ser domesticus_ | 1.40 | Series 1: Subcutaneous, 0.5, | | 1.0, 2.0, and 4.0 ml.

--------------------------+----------+--------------------------------- _Estrilda amandava_ | 0.45 | [A]Series 1: Intravenous, 0.5, | | 1.0, 2.0, and 4.0 ml.

| | | | [A]Series 2: Subcutaneous, 0.5, | | 1.0, and 2.0 ml.

| | | | Intraperitoneal, 8.0 ml.

--------------------------+----------+--------------------------------- _Poephila guttata_ | 0.56 | [A]Same as for _Estrilda_.

--------------------------+----------+--------------------------------- _Molothrus ater_ | 0.65 | Series 1: Intravenous and | | subcutaneous, respectively, 0.5 | | and 0.5 ml., 1.0 and 1.0 ml., | | 3.0 and 1.0 ml., 5.0 and 3.0 ml.

| | | | Series 2: Subcutaneous, 0.5, | | 1.0, 2.0 and 4.0 ml.

--------------------------+----------+--------------------------------- _Piranga rubra_ | 0.50 | Same as for _Molothrus_.

--------------------------+----------+--------------------------------- _Richmondena cardinalis_ | 0.70 | [A]Same as for _Estrilda_.

--------------------------+----------+--------------------------------- _Richmondena cardinalis_ | 0.60 | Same as for _Spinus_.

--------------------------+----------+--------------------------------- _Pa.s.serina cyanea_ | 0.45 | Antiserum not prepared.

--------------------------+----------+--------------------------------- _Spiza americana_ | 0.70 | Same as for _Molothrus_.

--------------------------+----------+--------------------------------- _Carpodacus purpureus_ | 0.50 | Antiserum not prepared.

--------------------------+----------+--------------------------------- _Spinus tristis_ | 0.49 | Series 1: Intravenous, 0.5, 1.0, | | 2.0, and 4.0 ml.

| | | | Series 2: Intravenous, 0.5, 1.0, | | 2.0, and 4.0 ml.

| | | | Series 3: Subcutaneous, 0.5, | | 1.0, 2.0, and 4.0 ml.

--------------------------+----------+--------------------------------- _Pipilo erythrophthalmus_ | 0.92 | Antiserum not prepared.

--------------------------+----------+--------------------------------- _Junco hyemalis_ | 0.56 | Same as for _Spinus_.

--------------------------+----------+--------------------------------- _Spizella arborea_ | 0.48 | Same as for _Spinus_.

--------------------------+----------+--------------------------------- _Zonotrichia querula_ | 0.48 | Same as for _Spinus_.

--------------------------+----------+--------------------------------- _Zonotrichia albicollis_ | 0.92 | Antiserum not prepared.

(Gmelin) | | --------------------------+----------+---------------------------------

[A] Antiserum prepared against formolized antigen.

Preparation of Antisera

All antisera were produced in rabbits (laboratory stock of _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 h.o.m.ologous 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 2C. 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 2C. 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 quant.i.tative specificities in their actions; therefore, when an antiserum containing precipitins is mixed with each of several antigens, the reaction involving the h.o.m.ologous 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 h.o.m.ologous 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 pa.s.sed 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 a.n.a.lyses of precipitin reactions. Numerals represent relative amounts of reaction between antigens and antisera. h.o.m.ologous 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 ------------------------+-----+-----+-----+-----+-----+-----+-----+---- _Pa.s.ser 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 2C. 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.