Part 7

The m. obturator externus is bipartite, consisting of dorsal and ventral parts, in the passerine species studied by Hudson (1937) and in all of the species examined by me except the ploceids and the cardueline finches. In the ploceids and cardueline finches this muscle is undivided and resembles in its position, origin, and insertion only the ventral portion of the muscle found in the other birds studied. It is difficult to imagine what advantage or disadvantage might be associated with the bipartite or with the undivided condition. The action of this muscle is to rotate the femur (right femur clockwise, left femur counterclockwise), and certainly the greater mass of the bipartite muscle could lend greater strength to such action. The possible significance of this is discussed below.

List of Abbreviations Used in Figures

Abd. dig. IV M. abductor digiti IV Acc. M. accessorius semitendinosi Add. long. M. adductor longus et brevis Anterolat. can. Anterolateral canal of hypotarsus Anteromed. can. Anteromedial canal of hypotarsus Bic. fem. M. biceps femoris Bic. loop Loop for m. biceps femoris Ext. cot. External cotyla Ext. dig. l. M. extensor digitorum longus Ext. hal. l. M. extensor hallucis longus Fem. tib. ext. M. femorotibialis externus Fem. tib. int. M. femorotibialis internus Fem. tib. med. M. femorotibialis medius F. dig. l. M. flexor digitorum longus F. hal. brev. M. flexor hallucis brevis F. hal. l. M. flexor hallucis longus F. p. et p. d. II M. flexor perforans et perforatus digiti II F. p. et p. d. III M. flexor perforans et perforatus digiti III F. per. d. II M. flexor perforatus digiti II F. per. d. III M. flexor perforatus digiti III F. per. d. IV M. flexor perforatus digiti IV Gas. M. gastrocnemius Iliacus M. iliacus Il. tib. M. iliotibialis Il. troc. ant. M. iliotrochantericus anticus Il. troc. med. M. iliotrochantericus medius Il. troc. post. M. iliotrochantericus posticus Int. cot. Internal cotyla Isch. fem. M. ischiofemoralis Midmed. can. Midmedial canal of hypotarsus Obt. ext. M. obturator externus Obt. int. M. obturator internus P. ant. Pars anticus P. ext. Pars externa P. int. Pars interna P. med. Pars media P. post. Pars posticus Per. brev. M. peroneus brevis Per. long. M. peroneus longus Pirif. M. piriformis Plan. M. plantaris Posterolat. can. Posterolateral canal of hypotarsus Posteromed. can. Posteromedial canal of hypotarsus Sar. M. sartorius Semim. M. semimembranosus Semit. M. semitendinosus Tib. ant. M. tibialis anticus Tib. cart. Tibial cartilage

The division of the pars interna of the m. gastrocnemius into anterior and posterior parts has not been reported by previous authors yet the division is quite distinct in those birds in which it occurs. Hudson (1937:36) points out that in some non-passerine birds the pars interna is double, but that in these species the m. semimembranosus inserts between the two parts. This is not the condition in those species studied by me. Only the ploceids and the cardueline finches in the present investigation fail to show such a division. The undivided muscle in these birds resembles, in its origin and position, the posterior portion of the muscle found in those species showing the bipartite condition. The greater mass of the bipartite muscle probably makes possible a stronger extension of the tarsometatarsus.

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 bipartite 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 bipartite 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 Passer, 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 Passer, in the estrildines, and in the carduelines probably is reduced.

In Passer, 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 quantitative nature, but within the past decade, satisfactory quantitative 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 quantitative 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 Cumley 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 2 deg.C., 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 2 deg.C. 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 2 deg.C. 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 2 deg.C. (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. --------------------------+----------+--------------------------------- Passer domesticus | 1.40 | Series 1: Subcutaneous, 0.5, | | 1.0, 2.0, and 4.0 ml. --------------------------+----------+--------------------------------- Estrilda amandava | 0.45 | Series 1: Intravenous, 0.5, | | 1.0, 2.0, and 4.0 ml. | | | | Series 2: Subcutaneous, 0.5, | | 1.0, and 2.0 ml. | | | | Intraperitoneal, 8.0 ml. --------------------------+----------+--------------------------------- Poephila guttata | 0.56 | 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 | Same as for Estrilda. --------------------------+----------+--------------------------------- Richmondena cardinalis | 0.60 | Same as for Spinus. --------------------------+----------+--------------------------------- Passerina 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) | | --------------------------+----------+---------------------------------

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 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.