The anatomy and histochemistry of flight hindlimb posture in birds. II. The flexed hindlimb posture of perching birds

Abstract

During flight, birds employ one of two hindlimb postures. Perching birds utilize a flexed posture with their folded legs tucked beneath the body, whereas shorebirds and raptors use an extended posture with straightened legs trailing behind the body. Maintenance of either posture during flight requires the hindlimbs to hold their position for prolonged periods. Slow contracting fibers are known for their fatigue‐resistant properties and are often found in high percentages in muscles utilized for postural actions. Given the similar actions required of the hip and knee flexors used during flight, we hypothesized that the equivalent postural muscles of perching birds (flexed posture) would contain similar percentages of slow fibers as shorebirds (extended posture). We investigated the anatomy and fiber type composition of seven hindlimb muscles in yellow‐headed and red‐winged blackbirds and revealed that they possess a smaller percentage of slow fibers than we found previously in the same muscles of American avocets and black‐necked stilts. The comparably smaller body size of yellow‐headed and red‐winged blackbirds could mitigate the need for more slow fibers. In addition, the biomechanical placement of the weight force in the flexed posture may require less muscle force for postural support during flight and, therefore, fewer slow fibers.

Introduction

During flight, birds can exhibit different postural behaviors. The forelimb may perform a variety of non‐flapping actions, including soaring and gliding, whereas the hindlimbs are typically held in place during most flight modes. One of two basic hindlimb postures is adopted: flexed or extended (Shepard et al. 2008). In the flexed posture, the hindlimbs are held beneath the body with the hip, knee, and ankle joints flexed. This posture is used by the majority of birds, including perching birds and woodpeckers. In the extended posture, the foot typically trails behind the body, with the hip and knee flexed, and the ankle extended. In general, this posture is used by shorebirds, waterfowl, and raptors (McFarland & Meyers, 2008). Maintaining either position requires that hindlimb muscles provide postural support against the force of gravity for periods ranging from brief flights to lengthy migrations.

Vertebrate skeletal muscle is generally composed of two broadly defined types of muscle fibers, fast and slow. These fibers vary in their contraction velocities, force production, aerobic capacity, and fatigability due to differences in their morphology and biochemistry (e.g. Hermanson, 1998; Hildebrand & Goslow, 1998; Pette & Staron, 2000). Fast fibers possess high contraction speeds and are often involved in more powerful movements such as running and flying. In contrast, slow muscle fibers are slower to contract and exhibit greater fatigue‐resistant properties. Slow fibers are often found in high percentages in muscles used for isometric contractions and postural behaviors (e.g. Rosser & George, 1986; Rosser et al. 1994; Hermanson, 1997).

In birds and mammals, one can observe a relationship between fiber type composition and muscle function. For example, in turkey vultures (Cathartes aura), white pelicans (Pelecanus erythrorhynchos), and albatrosses (Diomedeidae), the M. pectoralis is divided into a deep layer made up of slow fibers and a superficial layer composed of fast fibers (Rosser & George, 1986; Rosser et al. 1994; Meyers & Stakebake, 2005). In each of these birds, the slow‐fiber layer is presumed to hold the wing outstretched during soaring or gliding flight. In horses (Equus caballus), the M. extensor carpi radialis is functionally divided into a compartment of slow fibers, likely used for standing, and a separate population of fast fibers that can be used for locomotion (Hermanson, 1997).

Previous studies of avian hindlimb muscle function have mainly focused on terrestrial and aquatic locomotion (e.g. Gatesy, 1999; Clifton et al. 2018). However, McFarland & Meyers (2008) investigated the fiber type composition of muscles used for holding the hindlimbs of American avocets (Recurvirostra americana) and black‐necked stilts (Himantopus mexicanus) in the extended posture during flight. Their findings showed populations of slow fibers present in hindlimb muscles that they hypothesized to function in postural maintenance during flight. Furthermore, these muscles displayed distribution patterns of muscle fibers that were consistent across individuals. In light of their observations, our goals were to describe the hindlimb muscles involved in the flexed hindlimb posture, assay them for the presence of slow fibers, and quantify their respective percentages for comparison with those collected in the extended hindlimb study. We hypothesize that the hip and knee flexors of perching birds utilizing the flexed posture will show similar slow fiber percentages and distribution patterns to those found in avocets and stilts, as these muscles are performing the same action in both postures. In addition, we discuss the implications of the differences between the two postures.

Materials and methods

Anatomical and histochemical analysis was performed on four adult yellow‐headed blackbirds (Xanthocephalus xanthocephalus Bonaparte; three males and one female) and three adult red‐winged blackbirds (Agelaius phoeniceus L.; two males and one female). All birds were wild‐caught under appropriate collection permits issued to Dr. Franz Goller and euthanized in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Utah. The left leg of each bird was dissected and muscles removed for histochemical analysis within 1 h after death. The right leg and pelvis were left intact, fixed in 5% formalin, and stored in 1% phenoxyethanol for anatomical study. Skeletal material was obtained from the Smithsonian Institution National Museum of Natural History, Washington, DC, USA.

Muscles chosen for examination included the Mm. iliotibialis cranialis, iliotrochantericus caudalis, and iliotrochantericus cranialis, which are considered hip flexors; the Mm. iliofibularis, flexor cruris lateralis, and flexor cruris medialis, which are considered knee flexors; and the M. tibialis cranialis, the principal ankle flexor. These muscles were determined to function in the flexed hindlimb posture after manipulation of fresh specimens and evaluation of the literature (Raikow, 1985). Anatomical nomenclature is from Nomina Anatomica Avium (Baumel et al. 1993).

Dissection began by removing muscles of interest and cutting the middle of the muscle belly perpendicular to fascicle orientation. Procedures follow that described by McFarland & Meyers (2008). Muscle samples were mounted onto cork blocks using 5% gum tragacanth. Blocks were flash frozen in isopentane cooled in liquid nitrogen to approximately −150 °C and stored at −70 °C in an ultracold freezer. Serial sections 10‐μm‐thick, were cut using a freezing microtome and then transferred to microscope slides which were subsequently placed in a frozen coplin jar for transport.

Serial sections were reacted with S58 (DSHB, Iowa City, IA, USA), an antibody that binds to slow myosin heavy chains. Fibers that show a positive reaction with S58 are identified as slow muscle fibers. Fast fiber types were identified by reacting additional serial muscle sections with F18 (DSHB) an antibody that binds to fast myosin heavy chains (Fig. 1).

Figure 1.

Serial sections of M. iliotrochantericus caudalis from a yellow‐headed blackbird (Xanthocephalus xanthocephalus) showing representative reactions with the anti‐fast antibody (F18, top) and the anti‐slow antibody (S58, bottom). The fiber labeled ‘F’ reacted positively for the F18 antibody and negatively for the S58 antibody. The fiber labeled ‘S’ reacted positively for the S58 antibody and negatively for the F18 antibody. Scale bar: 50 μm.

Muscle cross‐sections were incubated in a humid chamber at room temperature for 2 h. They were rinsed with phosphate‐buffered saline solution (pH 7.2–7.4) and then underwent another (30‐min) incubation period, this time with anti‐mouse IgA antibody (Vector Laboratories, Burlingame, CA, USA) for slides reacted with the F18 antibody, and an anti‐goat antibody (Life Technologies, Carlsbad, CA, USA) for those reacted with S58. Slides were then developed and stained using an AEC peroxidase kit (Vector Laboratories). Muscle cross‐sections obtained from one of the yellow‐headed blackbirds were additionally reacted for the presence of myofibrillar ATPase in either acidic (pH 4.2) or alkaline (pH 10.35–10.45) preincubations to verify antibody data, using the protocol described in Meyers & Mathias (1997).

After the reactions were completed, slides were coverslipped using mounting gel and viewed under a Zeiss Axioskop 40 microscope (Oberkochen, Germany). A Canon Rebel T1i digital camera (Ota, Tokyo, Japan) was used to obtain overlapping pictures of each muscle cross‐section and their corresponding scale bar. The overlapping images were then merged and assembled using Adobe photoshop (San Jose, CA, USA) to create an image of the entire muscle cross‐section. Once the images were assembled, they were printed to allow for quantification of muscle fiber types.

Fast and slow muscle fibers were counted from either F18 or S58 images depending on which reaction provided the best contrast and visualization of fiber borders. Total counts for both fast and slow fiber types were obtained from full muscle cross‐sections from each available muscle for all specimens. Fiber type distribution patterns of each muscle from each individual were also recorded. Distribution patterns were qualitatively compared between individuals. Fiber type totals were entered into a table where respective percentages and standard deviations were calculated using Microsoft excel (Redmond, WA, USA).

Results

The hindlimb anatomy of yellow‐headed blackbirds is very similar to that of red‐winged blackbirds, which has been described previously by George & Berger (1966). For each muscle examined, a brief description is given, and the fiber type percentages are reported. Hybrid fibers (see Pette & Staron, 2001) are indicative of fiber type transitions and are marked by co‐reactivity to different antibodies. We did not observe any muscle fibers that reacted to both the S58 and F18 antibodies.

Hip flexion

M. iliotibialis cranialis

The M. iliotibialis cranialis (ITC) is a long, strap‐like muscle that extends from the cranial synsacrum to the medial tibiotarsus. ITC arises from the dorsal aponeurosis it shares with the M. rhomboideus profundus and from the cranial edge of the ilium. The muscle inserts onto the medial aspect of the proximal tibiotarsus, adjacent to the origin of the M. gastrocnemius pars internus (Fig. 2). The anatomy of this muscle was similar in both species.

Figure 2.

Right lateral view of the skeletal and muscular anatomy of the yellow‐headed blackbird (Xanthocephalus xanthocephalus) highlighting the muscles presumed to maintain the flexed hip during flight, in addition to a graphical representation of the slow fiber type percentage in each muscle: IlioCr, M. iliotrochantericus cranialis; IlioCa, M. iliotrochantericus caudalis; ITC, Iliotibialis cranialis; IF, iliofibularis; FCM, flexor cruris medialis; FCL, flexor cruris lateralis; TC, tibialis cranialis; fe, femur; fi, fibula; py, pygostyle; ti, tibiotarsus; tm, tarsometatarsus. Muscle cross‐sections show anti‐slow S58 antibody reactions where slow fibers are reacted positively and appear red; cranial is to the right of the page and lateral is to the top of the page. Scale bars: 1 mm, referring to the individual muscle cross‐sections only.

The M. iliotibialis cranialis showed varied fiber distributions amongst the six specimens. One yellow‐headed blackbird and two red‐winged blackbirds showed more similar distribution patterns, with slow fibers concentrated along the caudomedial edge of the muscle. One yellow‐headed blackbird had slow fibers evenly distributed in the caudal half of the muscle (Fig. 2) and in another all the fibers were fast fibers. The fourth individual possessed slow fibers interspersed with fast fibers in the cranial half of the muscle. Slow fibers composed 0.0−8.5% of the muscle in ITC for both species (Table 1).

Table 1.

Total fiber numbers, percentages of slow fibers, and mean percentage of slow fibers of hindlimb muscles from yellow‐headed blackbirds, Xanthocephalus xanthocephalus, and red‐winged blackbirds, Agelaius phoeniceus

Muscle	Yellow‐Headed Blackbirds					Red‐Winged Blackbirds
	Bird	# of slow fibers	# of fast fibers	% of slow fibers	Mean % slow fibers + SD	Bird	# of slow fibers	# of fast fibers	% slow fibers	Mean % slow fibers ± SD
M. iliotibialis cranialis	1 F	336	3619	8.5%	4.4 ± 3.9%	1 M	257	3460	6.9%	6.2 ± 1.1%
	2 M	195	2665	6.8%		2 M	255	4468	5.4%
	3 M	93	4094	2.2%
	4 M	0	922	0.0%
M. iliotrochantericus caudalis	1 F	983	6855	12.5%	11.7 ± 2.7%	1 M	623	4340	12.6%	11.8 ± 1.0%
	2 M	676	7576	8.2%		2 M	438	3522	11.0%
	3 M	1168	9265	11.2%
	4 M	1671	9662	14.7%
M. iliotrochantericus cranialis	1 F	93	2214	4.0%	2.9 ± 0.8%	1 M	45	2872	1.5%	1.5
	2 M	90	3269	2.7%
	3 M	97	4172	2.3%
	4 M	46	1661	2.7%
M. iliofibularis	1 F	753	4606	14.1%	12.2 ± 2.3%	1 M	827	5760	12.6%	8.8 ± 3.7%
	2 M	1178	7360	13.8%		2 M	310	5613	5.2%
	3 M	1037	10322	9.1%		3 F	627	6589	8.7%
	4 M	948	6962	12.0%
M. flexor cruris lateralis	1 F	0	4717	0.0%	0.0%	1 M	0	4495	0.0%	0.0%
	2 M	0	6122	0.0%		2 M	0	3667	0.0%
	3 M	0	6400	0.0%		3 F	0	4357	0.0%
	4 M	0	4308	0.0%
M. flexor cruris medialis	1 F	212	2213	8.7%	7.0 ± 2.0%	1 M	503	3969	11.2%	11.4 ± 2.6%
	2 M	108	2337	4.4%		2 M	335	3463	8.8%
	3 M	447	4923	8.3%		3 F	494	3008	14.1%
	4 M	206	2997	6.4%
M. tibialis cranialis	1 F	1443	7560	16.0%	17.0 ± 1.0%
	2 M	1243	5548	18.3%		2 M	435	2984	12.7%	13.7 ± 1.4%
	3 M	1975	10047	16.4%		3 F	1068	6194	14.7%
	4 M	1533	7460	17.0%

M. iliotrochantericus caudalis

The M. iliotrochantericus caudalis (IlioCa) is a large triangular muscle located on the cranial aspect of the synscacrum, where it occupies most of the iliac fossa. IlioCa arises from the rim and surface of the iliac fossa of the synsacrum, and tapers as it approaches the proximal femur. It inserts on the lateral aspect of the proximal femur, just distal to the trochanter, proximal to the insertion of the M. iliotrochantericus cranialis (Fig. 2). The anatomy of IlioCa was the same for both blackbirds.

In three of the four yellow‐headed blackbirds, slow fibers were heavily concentrated at the caudal tip of the muscle and became uniformly distributed on the medial side of the central tendon. The cranial end was composed completely of fast fibers (Fig. 2). A fourth individual did not have a concentration of slow fibers at the caudal edge but slow fibers were evenly distributed. In both of our red‐winged blackbird specimens, IlioCa also possessed slow fibers at the caudal end of the muscle, becoming uniformly distributed with fast fibers on the lateral side of the central tendon. Slow fibers composed 8.2−14.7% of the muscle in both blackbird species. (Table 1).

M. iliotrochantericus cranialis

The M. iliotrochantericus cranialis (IlioCr) is a small, strap‐like muscle that extends from the cranial synsacrum to the proximal femur. IlioCr arises from the cranial edge of the iliac fossa of the synsacrum. In one yellow‐headed blackbird individual, the muscle also originated from adjacent fascicles of the M. iliotrochantericus caudalis. IlioCr inserts onto the proximal femur, distal to the insertion of the M. iliotrochantericus caudalis (Fig. 2). This muscle had the same morphology in both blackbird species.

In three of the four yellow‐headed blackbirds, a small population of slow fibers was interspersed with fast fibers and located near the caudomedial edge of IlioCr. The lateral half of the muscle was uniformly fast. One of the yellow‐headed blackbirds had slow fibers that were concentrated toward the caudal half along the midline of the muscle. IlioCr from one red‐winged blackbird possessed a small number of slow fibers distributed along the medial edge of the muscle. Overall, slow fibers made up 1.5–4.0% of the muscle in all individuals (Table 1).

Knee flexion

M. iliofibularis

The M. iliofibularis (IF) is roughly triangular in shape and extends from the ilium to the fibula. It arises from the lateral surface of the caudal iliac crest of the synsacrum and tapers as it approaches the tibiotarsus. The muscle forms a thick tendon, which passes through a fibrous loop (the Ansa m. iliofibularis) and is redirected distally along the fibula, where it attaches onto a tubercle on the lateral aspect of that bone (Fig. 3). The anatomy of IF was similar in both blackbird species.

Figure 3.

Right lateral view of the skeletal and muscular anatomy of the yellow‐headed blackbird (Xanthocephalus xanthocephalus) highlighting the muscles presumed to maintain the flexed knee during flight, in addition to a graphical representation of the slow fiber type percentage in each muscle: IF, iliofibularis; FCM, flexor cruris medialis; FCL, flexor cruris lateralis; IlioCr, M. iliotrochantericus cranialis; IlioCa, M. iliotrochantericus caudalis; ITC, iliotibialis cranialis; TC, tibialis cranialis; fe, femur; fi, fibula; py, pygostyle; ti, tibiotarsus; tm, tarsometatarsus. Muscle cross‐sections show anti‐slow S58 antibody reactions where slow fibers reacted positively and appear red; cranial is to the right of the page and lateral is toward the top of the page. Scale bars: 0.5 mm, referring to individual muscle cross‐sections only.

The fiber type distribution pattern for IF was consistent among all yellow‐headed and red‐winged blackbird specimens. Slow fibers were uniformly distributed with fast fibers from the cranial edge throughout the first two‐thirds to three‐fourths of the muscle. The caudal end contained only fast fibers (Fig. 3). Slow fibers made up 5.2–14.1% of IF (Table 1).

M. flexor cruris lateralis

The M. flexor cruris lateralis (FCL) is a strap‐like muscle that extends from the ilium to the caudal femur and lies between the M. iliofibularis laterally and the M. flexor cruris medialis medially. FCL pars pelvica takes origin from the lateral surface of the iliac crest, caudal to the M. iliofibularis, and from an aponeurosis extending from the caudal ilium to the proximal caudal vertebrae. The muscle runs towards the crus, where after about three‐fourths of its length, a tendinous raphe divides FCL. This raphe is strongly attached to the tendinous origin of the M. gastrocnemius pars intermedia and has the effect of connecting FCL to that muscle. The muscle continues from the raphe as ‘pars accessoria’ and inserts onto the caudal aspect of the distal femur (Fig. 3). Comparison reveals that in red‐winged blackbirds, the raphe extends as an aponeurosis and inserts with FCM onto the proximal caudomedial shaft of the tibiotarsus, as also described by George & Berger (1966). FCL was completely fast in all individuals of both species (Fig. 3). Slow fibers made up 0.0% of the muscle (Table 1).

M. flexor cruris medialis

The M. flexor cruris medialis (FCM) is the thinnest, most strap‐like of the three crural flexors and lies medially and mostly deep to FCL. FCM arises from the lateral surface of the ischium, around the fenestra ischiopubica. It maintains a parallel shape and inserts via a thin aponeurosis onto the medial aspect of the proximal tibiotarsus, deep to the M. gastrocnemius pars medialis (Fig. 3). FCM was similar in both blackbird species.

Flexor cruris medialis presented a fiber type distribution pattern that was consistent across all individuals of both species. Slow fibers were uniformly distributed with fast fibers along the medial half of the muscle starting at the cranial edge. Slow fibers decreased in number towards the caudal edge, which was completely composed of fast fibers (Fig. 3). Slow fibers made up 4.4–14.1% of the muscle (Table 1).

Ankle flexion

M. tibialis cranialis

The M. tibialis cranialis (TC) occupies the cranial aspect of the tibiotarsus, where it is laterally largely deep to the M. fibularis longus. TC arises by two robust heads: A smaller femoral head takes origin by a short tendon from a small pit (the Fovea tendineus m. tibialis cranialis) on the lateral condyle of the femur, and a larger tibial head arises from the cnemial crest of the tibiotarsus. About four‐fifths down the length of the tibiotarsus, the two heads merge to form a single belly that inserts by a stout tendon onto the Tuberosity m. tibialis cranialis on the proximal tarsometatarsus (Fig. 4). Comparison of the two species indicates that the fusion of the heads in red‐winged blackbirds occurs more proximally than in yellow‐headed blackbirds.

Figure 4.

Right lateral view of the skeletal and muscular anatomy of the yellow‐headed blackbird (Xanthocephalus xanthocephalus) highlighting the ankle flexor presumed to maintain the flexed ankle during flight, in addition to a graphical representation of the slow fiber type percentage for the muscle: TC, tibialis cranialis; IF, iliofibularis; FCM, flexor cruris medialis; FCL, flexor cruris lateralis; IlioCr, M. iliotrochantericus cranialis; IlioCa, M. iliotrochantericus caudalis; ITC, iliotibialis cranialis; fe, femur; fi, fibula; py, pygostyle; ti, tibiotarsus; tm, tarsometatarsus. Muscle cross‐sections show anti‐slow S58 antibody reactions where slow fibers reacted positively and appear red; cranial is to the right of the page and lateral is to the top of the page. Scale bar: 1 mm, referring to the muscle cross‐section only.

In all yellow‐headed blackbirds, slow fibers were uniformly distributed in the tibial head of TC. The femoral head showed more variability in its distribution pattern, but it consistently possessed fewer slow fibers when compared with the tibial head (Fig. 4). In red‐winged blackbirds, slow fibers were more variably distributed within both the tibial and femoral heads. Overall, slow fibers ranged from 12.7 to 18.3% of the TC in blackbirds (Table 1).

Discussion

Birds display two different hindlimb flight postures, flexed and extended. Perching birds, such as blackbirds, utilize the flexed hindlimb flight posture. In this posture, the hindlimbs are held tucked beneath the body with the hip, knee, and ankle in a flexed position. In contrast, shorebirds, such as avocets and stilts, use the extended posture, with the hip and knee flexed, and the ankle extended. Why some birds use one posture or another is unknown at present, but this feature appears taxonomically stable. More primitive avian groups (e.g. Anseriformes) use the extended posture and more derived ones (e.g. Passeriformes) use the flexed posture (taxonomy from Jarvis et al. 2014). Barrett‐Hamilton (1903) noted the groups of birds that make use of the two postures and attributed the postures to limb segment length and foot size. Shepard et al. (2008) suggested various explanations, including differences in aerodynamics, morphology, and evolutionary history, but a conclusion is difficult to reach and warrants further investigation.

We hypothesized that the hip and knee flexors of blackbirds would contain similar slow fiber percentages and distribution patterns as those found in the hip and knee flexors of shorebirds, because these muscles are performing the same action in both postures. Contrary to our hypothesis, the slow fiber type composition within both the hip and knee flexors in blackbirds was different than that found in shorebirds. In comparison with data collected for avocets and stilts, the hindlimb muscles of both blackbirds contained fewer slow fibers and more variable distribution patterns.

Muscles involved in flexed hindlimb posture

Hip flexion

Previous analysis of the M. iliotibialis cranialis (ITC) in the wild common coot (Fulica atra) by Torrella et al. (1998a) found that the caudal portion contained an average of 22–26% slow fibers. Torrella et al. (1996) reported 17–19% slow fibers in the caudal ITC and 2% in the cranial ITC of the mallard (Anas platyrhynchos). In the yellow‐legged gull (Larus michahellis), Torrella et al. (1998b) reported an average of 14–16% slow fiber type in the caudal ITC and 6% in the cranial portion. In addition, Suzuki et al. (1985) described 27% slow fibers in the medial region of ITC in chickens (Gallus gallus), with less than 1% slow fibers in the lateral region. McFarland & Meyers (2008) reported 12–19% slow fibers in the ITC of avocets and stilts. They described slow fibers being interspersed with fast fibers in the medial half of the muscle. In contrast, we found fewer (0.0–8.5%) slow fibers in the ITC of both species of blackbirds, with variable distribution patterns amongst individuals. The blackbird percentages are substantially lower than the other species examined, all of which have a comparatively greater body mass.

To date, there are few data available regarding the fiber type composition of the M. iliotrochantericus caudalis (IlioCa) and the M. iliotrochantericus cranialis (IlioCr). McFarland & Meyers (2008) reported that slow fibers in IlioCa ranged from 30 to 48% in avocets and stilts. In all individuals, IlioCa has a distinct fiber type distribution pattern; the lateral half of the muscle is completely slow, and the medial half has a mixed fast−slow fiber composition. In comparison, the IlioCa in both blackbird species possessed 8.2–14.7% slow fibers. IlioCa consistently had a pocket of slow fibers at the caudal end of the muscle (Fig. 2). However, both species of blackbirds show some variation in distribution pattern, as the slow fibers gradually become mixed with fast fibers on either the medial or lateral side of the internal tendon. Given the complex architecture of this muscle, it likely has many possible actions, including protraction, medial rotation and adduction, and retraction of the hindlimb (see Raikow, 1985). Electromyographical studies may reveal any functional compartments in this muscle that may function during different behaviors.

Within the IlioCr of avocets and stilts, McFarland & Meyers (2008) described 8–16% slow fibers evenly distributed with fast fibers throughout the muscle, except for the cranial region. In the IlioCr of both blackbirds, slow fibers only made up 1.5–4.0% of the muscle. In all four yellow‐headed and one red‐winged blackbird, slow fibers were mixed with fast fibers caudomedially (Fig. 2).

Knee flexion

There have been few investigations of the muscle fiber types of the knee flexors of birds. Cracraft (1971) described the M. iliofibularis (IF) in pigeons (Columba livia) as having fewer than 20% slow tonic fibers. In addition, Suzuki et al. (1985) reported 20–21% slow fibers in the cranial, caudomedial, and caudolateral regions of IF in chickens. They also described 7% slow fibers in the middle and medial region of the muscle, which is comparable in percentage and location to our results of IF. McFarland & Meyers (2008) reported 15–25% slow fibers mixed with fast fibers within the medial half of IF in avocets and stilts. Very few slow fibers were found in the lateral half. In both species of blackbirds, slow fibers were evenly distributed with fast fibers up until the caudal end of the muscle, at which point the muscle is made up completely of fast fibers (Fig. 3). Slow fibers made up a smaller percentage of these muscles in perching birds, with 9.1–14.1% in the IF of four yellow‐headed blackbirds and 5.2–12.6% in three red‐winged blackbirds. As was the case with hip flexors, blackbirds knee flexors possess lower percentages of slow fibers than do the species of higher body mass previously studied.

Previous studies on the fiber type composition of the M. flexor cruris lateralis report contrasting results. Cracraft (1971) described FCL as having a ‘mixed’ composition in pigeons. However, a study on the locomotor muscles of tufted ducks (Aythya fuligula) revealed that the FCL did not possess any slow fibers (Turner & Butler, 1988). Previous work on avocets and stilts show that the FCL had 2–11% slow fibers distributed along the craniolateral edge of the muscle (McFarland & Meyers, 2008). Our results were similar to those of the tufted duck in that all of the fibers of FCLs examined within both species of blackbirds were completely fast (Fig. 3).

Although FCL is connected to the M. gastrocnemius via the raphe, its insertion is onto the distal femur, which would make it a hip extensor (retractor), not a knee flexor. The absence of slow fibers would support this idea that FCL is not involved in holding the limbs during flight, a hypothesis testable with electromyography. The complex nature of the raphe's connection to other structures in the leg (see Raikow, 1985) would suggest that the role this muscle plays in flight posture could be variable in different species.

In the M. flexor cruris medialis, Cracraft (1971) reported that many of the fibers may be slow tonic in pigeons. Suzuki et al. (1985) found that the medial region of the chicken FCM contained 21% slow fibers, whereas the lateral region only contained 1% slow fibers. McFarland & Meyers (2008) revealed that slow fibers composed 10–25% of FCM and were distributed with fast fibers within the medial portion of the muscle in avocets and stilts. We found that the FCM of both blackbird species contained 4.4–14.1% slow fibers. The fiber type distribution pattern was similar to that of avocets, with slow fibers being interspersed amongst fast fibers along the medial half of the muscle. However, unlike avocets and stilts, slow fibers were absent near the cranial edge of FCM in both species of blackbirds (Fig. 3).

Ankle flexion

Very little is known about the fiber type composition of the M. tibialis cranialis in birds. Cracraft (1971) described it as a mixed muscle in pigeons, where fast twitch fibers are more numerous than slow tonic fibers. TC is a mixed muscle in both blackbird species as well. In yellow‐headed and red‐winged blackbirds, slow fibers comprised 12.7–18.3% of TC and were evenly mixed with fast fibers in the tibial head of the muscle (Fig. 4). The femoral head showed more variable distribution with fewer slow fibers. This fiber type distribution was more consistent in yellow‐headed blackbirds than in red‐winged blackbird specimens.

Patterns of muscle variation

As previously described by McFarland & Meyers (2008), the majority of hindlimb muscles in avocets and stilts possessed consistent slow fiber type distribution patterns, with little variation present among individuals and species. In blackbirds, we report a similar level of consistency in slow fiber distribution pattern between individuals for the flexors of the knee and ankle (Table 1). In contrast, the hip flexors of both blackbird species showed a greater degree of variation between individuals in the distribution of slow fibers. In the M. iliotibialis cranialis, four individuals had a similar distribution pattern where slow fibers were more concentrated along the caudomedial edge of the muscle. One individual lacked slow fibers entirely, and the remaining specimen possessed a distribution pattern that was the opposite, with slow fibers in the cranial region. Similarly, the distribution pattern of M. iliotrochantericus cranialis was fairly consistent among three individuals, with slow fibers concentrated along the caudomedial edge of the muscle; two other individuals showed different patterns.

We were further surprised by the absence of any slow fibers in the M. iliotibialis cranialis of one individual, whereas all of the other individuals possessed slow fibers. Although we would like to think that individual variation in fiber type percentage should be low, that does not seem to be the case. Wang & Kernell (2001) described individual variation in the tibialis anterior and extensor digitorum muscles of mice and the soleus muscle of rabbits. Although the mouse M. tibialis anterior averaged 5.5 slow fibers, one of their seven individuals had no slow fibers. Likewise, the M. extensor digitorum averaged 13 slow fibers, but two of seven individuals had no slow fibers. Two of six rabbit soleus muscles had no fast fibers. In one specific region of six horse biceps brachii muscles, Hermanson & Hurley (1990) reported a wide range of slow fiber percentages, ranging from 11 to 63%. It is unclear why this variation exists or what the functional ramifications may be, but Meyers & McFarland (2016) have discussed that in fast muscles, slow fibers are more plentiful during earlier developmental stages and that they are typically replaced by faster fibers. Variations in that developmental program could alter final fiber type ratios.

Muscle fiber type proportions are known to vary with sex (Haizlip et al. 2015), which have been attributed to differences in levels of male sex hormones. For example, different muscle fiber types have been observed in male and female forelimb muscles of toads (Bufo japonicus) (Oka et al. 1984), in laryngeal muscles of Xenopus (Sassoon et al. 1987), and in syringeal muscles in zebra finches (Taeniopygia guttata) (Christensen et al. 2017); in each of these examples, the muscles are used dimorphically between the sexes and testosterone is considered to be the driving force in the disparity. Similarly, androgens are implicated in the enhanced muscle contraction speed seen in wing‐snap behavior of golden‐collared manakins (Manacus vitellinus) (Fuxjager et al. 2017). Fiber type differences have also been reported in the masseter muscle of rabbits (English et al. 1999) and mice (Eason et al. 2000), and the temporalis muscle of guinea pigs (Lyons et al. 1986). In these cases, androgens are implicated as well, but no clear functional explanation was given. In our study, some sexual dimorphism in fiber percentages was observed, in which the female yellow‐headed blackbird had a greater percentage of slow fibers than males in four of the six muscles that had slow fibers, and in which the female red‐winged blackbird had a greater percentage in two of the three muscles containing slow fibers (see Table 1). Although male yellow‐headed blackbirds can be twice as large as females (Twedt & Crawford, 1995), we only have body mass measurements for two individuals examined in our study, one male and one female. The female weighed 102 g and one male (#4) 91 g. However, since we only examined a single female of both blackbird species, the extent of any dimorphism in size or sex cannot really be resolved. It is possible that given a larger survey of individuals, these differences would be enhanced or reduced.

It is widely reported that slow fibers are more commonly located in deeper regions of muscles in mammals (see Collatos et al. 1977; Acosta & Roy, 1987, and references therein) and birds (e.g. Turner & Butler, 1988; Torrella et al. 1996). In contrast, the shorebird muscles that McFarland & Meyers (2008) studied possessed superficially located slow fibers. Our results are consistent with their observations in that slow fibers were not restricted to deep regions of the muscles. Although hindlimb muscles can have many actions, we believe that this fiber distribution pattern relates to the biomechanical function of these muscles in hindlimb posture. Biomechanical advantage is one of a number of possible functions of slow fiber regionalization, according to Wang & Kernell (2001).

Body size implications

Body size has been shown to be a driver of differences in morphology and physiology for a variety of animals. For example, larger animals are more influenced by gravity and require modifications to their skeletons, muscles, and circulatory systems (McMahon & Bonner, 1983; Schmidt‐Nielsen, 1993; Vogel, 2003). A possible explanation for the observed differences in hindlimb fiber type composition is that avocets and stilts are much larger than blackbirds. Slow contracting fibers are known to be especially important in anti‐gravity muscles (Hermanson & Hurley, 1990; Suzuki, 1990; Savolainen & Vornanen, 1995), and smaller animals typically have fewer slow fibers in postural roles. According to Suzuki (1990) and Jürgens (2002), smaller/lighter animals have a reduced need for slow postural fibers because the effects of gravity on lighter animals are lower than on larger/heavier animals. For example, shrews have no slow fibers in any of their muscles that have been studied (Suzuki, 1990; Savolainen & Vornanen, 1995; Peters et al. 1999; Jürgens, 2002). Ariano et al. (1973) showed that larger mammals have greater percentages of slow muscle fibers in the quadriceps femoris and triceps surae muscle groups, which have known postural functions in mammals. Therefore, a possible explanation for the observed differences in hindlimb fiber type composition is that avocets and stilts are much larger than blackbirds.

Yellow‐headed blackbirds can weigh between 51 and 100 g (Twedt & Crawford, 1995), whereas American avocets weigh anywhere from 278 to 378 g (Ackerman et al. 2013). Thus, yellow‐headed blackbirds are three to four times lighter than avocets. We found that this difference in size is also reflected in the percentage of slow fibers in their hindlimb muscles; yellow‐headed blackbirds have one‐third to one‐fourth the percentage of slow fibers in hip flexors compared with avocets and stilts. For knee flexors, yellow‐headed blackbirds also had consistently lower slow fiber percentages than avocets and stilts. Our results, combined with those of other studies, support the observations that smaller animals typically have a lower percentage of slow muscle fibers in their postural muscles than larger animals do.

Biomechanics

Another possible explanation to account for the lower percentage of slow fibers in blackbirds may be the biomechanical differences between the two flight postures. The position of the hindlimbs in each posture creates a lever system in which equilibrium is necessary to hold the limbs in place (Hildebrand & Goslow, 1998). Consider the knee flexors in the extended posture (Fig. 5A). The in‐lever is the distance from the knee joint to the insertion of the muscle and is roughly the same length for both postures. The out‐lever is the distance from the knee joint to the position of the weight force of the hindlimbs. The amount of force that the muscle will need to exert is dependent upon the location of the weight force of the hindlimbs and, therefore, the limb posture.

Figure 5.

Anatomical representation and force diagram of a knee flexor in the extended (A – black‐necked stilt, Hemantopus mexicanus) and flexed (B – yellow‐headed blackbird, Xanthocephalus xanthocephalus) flight postures, drawn to scale. The toes are omitted in both. Each posture creates a lever system with an in‐lever (Li) and an out‐lever (Lo). The in‐lever is the distance from the knee joint to the insertion of the muscle (M), which is contracting isometrically to pull the tibiotarsus dorsally (blue arrow). The out‐lever is the distance from the knee joint to the position of the weight force (W) of the hindlimb (red arrow). Based on the equation M × Li = W × Lo, to hold the limbs in place, a muscle must exert an isometric force (muscle force, M) to counteract the weight force created by the hindlimb. As shown, the out‐lever of the flexed posture is considerably smaller than that of the extended posture and should require less muscle force to maintain equilibrium. (C) Illustrates the yellow‐headed blackbird limb modified into the extended posture to show that even with the same limb proportions, the out‐lever would be greater, still necessitating a greater muscle force. Scale bar: 1 cm, referring to both postures.

In the extended posture, the weight force of the hindlimbs is applied farther away from the bird's center of mass (to the left on Fig. 5A). This feature creates a larger out‐lever, which in turn increases the amount of force required of the knee flexors to maintain this posture during flight. However, in the flexed posture, the weight force of the hindlimbs is positioned closer to the bird's center of mass (Fig. 5B). As a result, the out‐lever in the flexed position is shorter than the out‐lever in the extended posture. If the out‐lever is shorter, less muscle force is required of the knee flexors to hold the joint stable during flight. Less muscle force would require fewer fatigue‐resistant, slow fibers for the maintenance of the flexed posture. Given a hypothetical bird with the dimensions of Fig. 5B but drawn using the extended posture, we can see the effects of this posture on the out‐lever distance (Fig. 5C), which is made more severe by the very elongated tibiotarsus of many shorebirds. However, determining the degree that limb posture influences slow fiber percentages would require a comparison between different species of the same body mass that utilize each hindlimb posture.

Our lab recently began an EMG study of the hindlimb postural muscles in yellow‐headed blackbirds. We believe that this avenue of research can elucidate how these muscles are being used during flight. In addition, investigation involving birds of comparable masses that utilize the two different postures is warranted to determine more accurately the influence of biomechanical differences on slow fiber percentage variation.

Based on McFarland & Meyers’ (2008) study of the muscles involved in the extended limb posture of shorebirds, we expected to find comparable muscle fiber type ratios in equivalent muscles of perching birds that use the flexed posture. We were surprised that blackbird limb muscles had much lower percentages of slow muscle fibers, but relate this difference to body size issues. The biomechanics of limb postures likely also play a role.

Author contributions

Conceived and designed the study: R.A.M. Performed the histology: A.M.W. Analyzed the data and wrote the manuscript: A.M.W., R.A.M.