posterior to the _m. flexor digitorum longus_, the belly of this
large, pinnate muscle is intimately connected anteriorly to that of
the _m. flexor perforatus digiti II_. The _m. flexor hallucis longus_
arises by two heads which are separated by the tendon of insertion of
the _m. biceps femoris_. The smaller anterior head arises from the
same tendon as does the _m. flexor perforatus digiti II_. The larger
posterior head arises by means of fleshy fibers from the
intercondyloid region of the posterior surface of the femur along with
the _m. flexor perforatus digiti III_ and _IV_. The two heads join
just distal to the point of insertion of the _m. biceps femoris_.
There is no trace of a tendinous band connecting the two heads as
there is in the crow and in the raven (Hudson, 1937:49). Near the
distal end of the shank the muscle gives rise to a strong tendon which
perforates the tibial cartilage along its lateral edge and passes
through the anterolateral canal of the hypotarsus (Fig. 6). The tendon
crosses over to the medial surface of the tarsometatarsus, passes
distally, and perforates the sheathlike tendon of the _m. flexor
hallucis brevis_ between the first metatarsal and the trochlea for
digit II. The tendon continues along the posterior surface of the
hallux and has a double insertion; the main tendon attaches to the
base of the ungual phalanx and a smaller branch inserts on the distal
end of the proximal phalanx.
Action.--Flexes hallux.
Comparison.--In _Vireo_ this muscle has only the posterior head of
origin and is not connected with the _m. flexor perforatus digiti II_.
The muscle is proportionately smaller and weaker than in any of the
other species studied.
_+Musculus extensor hallucis longus+_ (Fig. 4).--One of the smallest
muscles of the leg, the origin is fleshy from the anteromedial edge of
the proximal end of the tarsometatarsus. The belly is long and slender
and terminates distally in a slender tendon which passes distally
along the posterior surfaces of the first metatarsal and the first
digit. The insertion is on the base of the ungual phalanx. Near the
distal end of the proximal phalanx, the tendon passes between two
thick bands of fibro-elastic tissue which insert also on the ungual
phalanx. These bands of tissue function as automatic extensors of the
claw.
Action.--Extends hallux; action must be slight.
Comparison.--In _Vireo_ this muscle is proportionately larger and
better developed than it is in any of the other species examined.
_+Musculus flexor hallucis brevis+_ (Fig. 4).--This minute muscle has
a fleshy origin from the medial surface of the hypotarsus. The short
belly terminates in a weak, slender tendon which passes down the
posteromedial surface of the tarsometatarsus and into the space
between the first metatarsal and the trochlea for digit II. In this
region the tendon envelops the tendon of the _m. flexor hallucis
longus_ and inserts on the distal end of the first metatarsal and on
the proximal end of the first phalanx of the first digit.
Action.--Flexes hallux; action must be slight.
Comparison.--The small size of this muscle makes it exceedingly
difficult to study. The muscle is larger in _Vireo_ than in any of the
other species examined. This may be correlated with the smaller size
of the _m. flexor hallucis longus_ in this species. The muscle does
not seem to be so well developed in the cardueline finches as it is in
the other species.
_+Musculus abductor digiti IV+_ (Fig. 2).--Extremely small, delicate
and difficult to demonstrate, this muscle arises in a fleshy origin
immediately from underneath the posterior edge of the external cotyla
of the tarsometatarsus. The tendon of insertion is long and slender
and inserts along the lateral edge of the first phalanx of digit IV.
Action.--Abducts digit IV.
Comparison.--No significant differences noted among the species
studied.
_+Musculus lumbricalis.+_--Semitendinous throughout its length, this
muscle arises from the ossified tendon of the _m. flexor digitorum
longus_ at a point immediately proximal to the branching of this
tendon. The insertion is on the joint pulleys and capsules at the base
of the third and fourth digits.
Action.--Hudson (1937:57) states that: "Meckel (_vide_ Gadow--1891, p.
204) considered this muscle as serving to draw the joint pulley behind
in order to protect it from pinching during the bending of the toes.
It perhaps also tends to flex the third and fourth digits."
Comparison.--No significant differences noted among the species
studied.
Discussion of the Myological Investigations
Simpson (1944:12) and others have emphasized that different parts of
organisms evolve at different rates. Beecher (1951b:275) in stating
that "... the hind limb is very similar in muscle pattern throughout
the Order Passeriformes and seems to have become relatively static
after attaining a high level of general efficiency ..." implies that
the muscle pattern of the leg must be one of long standing and slow
change. This concept was emphasized by Hudson (1937) who found but
little variation in muscle pattern among members of the several
families of passerine birds. The concept is further confirmed by the
present investigation. The intricate patterns of origin and of
insertion seem to remain almost the same throughout the order in spite
of adaptive radiation which has occurred.
Two major differences in patterns of leg-musculature, however, were
found among the species studied, and these differences are significant
since they are consistent between subfamilies. The muscles involved
are the _m. obturator externus_ and the _pars interna_ of the _m.
gastrocnemius_.
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
[Illustration: FIG. 1. _Pipilo erythrophthalmus._ Lateral view of
the superficial muscles of the left leg, x 1.5.]
[Illustration: FIG. 2. _Pipilo erythrophthalmus._ Lateral view of
the left leg showing a deeper set of muscles. The superficial
muscles _iliotibialis_, _sartorius_, _gastrocnemius_ and
_peroneus longus_ have been removed, x 1.5.]
[Illustration: FIG. 3. _Pipilo erythrophthalmus._ Lateral view of
the left leg showing the still deeper muscles. In addition to
those listed for figure 2, the following muscles have been
wholly or partly removed: _iliotrochantericus posticus_,
_femorotibialis externus_, _femorotibialis medius_,
_biceps femoris_, _semitendinosus_, _tibialis anticus_,
_flexor perforans et perforatus digiti II_, and _flexor
perforans et perforatus digiti III_, x 1.5.]
[Illustration: FIG. 4. _Pipilo erythrophthalmus._ Medial view of
the superficial muscles of the left leg, x 1.5.]
[Illustration: FIG. 5. _Pipilo erythrophthalmus._ Medial view of
the left leg showing a deeper set of muscles than those seen
in figure 4. The following superficial muscles have been
removed: _iliotibialis_, _sartorius_, _femorotibialis internus_,
_obturator internus_, _adductor longus (pars posticus)_,
_gastrocnemius_, and _peroneus longus_, x 1.5.]
[Illustration: FIG. 6. _Pipilo erythrophthalmus._ Proximal end of
left tarsometatarsus and the hypotarsus, x 4.]
[Illustration: FIG. 7. _Pipilo erythrophthalmus._ Lateral view of
proximal end of left femur and a portion of the pelvis, x 3.5.]
[Illustration: FIG. 8. _Pipilo erythrophthalmus._ Upper surfaces
of the phalanges of the foretoes of the left foot showing
insertions of the _M. extensor digitorum longus_, x 3.]
[Illustration: FIG. 9. _Pipilo erythrophthalmus._ Medial view of
the second digit of the left foot, showing insertions of the
flexor muscles, x 3.]
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 | [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_.
--------------------------+----------+---------------------------------
_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) | |
--------------------------+----------+---------------------------------
[A] Antiserum prepared against formolized antigen.
Preparation of Antisera