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. 2026 Aug 13;55(5):e70165. doi: 10.1111/ahe.70165

Distinctive Innervation Patterns of Thigh Extensor Muscles in Anura: A Morphogenetic Hypothesis of Nerve Re‐Routing Within the Hindlimb Plexus in the Tetrapod Evolutionary Process

Hidaka Anetai 1,2,✉, Masaki Waki 2, Masaya Ohishi 2, Saori Anetai 2, Takashi Amari 2, Ryuhei Kojima 2, Kounosuke Tokita 2,✉
PMCID: PMC13473813  PMID: 42596606

ABSTRACT

In mammals, thigh muscles are compartmentalized into three groups: extensor, flexor, and adductor muscles, and these are innervated by the femoral nerve, sciatic nerve (tibial and common peroneal nerve divisions), and obturator nerve, respectively. However, amphibians show a different pattern of innervation. In this study, the extensor and flexor muscles, their innervation, and relation to the muscle compartment (by the lateral femoral intermuscular septum) were examined and noted in bullfrog thighs. Ten thighs of five bullfrog specimens were examined macroscopically and partially histologically. The extensor and flexor muscles were identified, and these two muscle groups were separated by the lateral femoral intermuscular septum in the dorsal thigh of bullfrogs. However, the nerve from the common peroneal nerve division of the sciatic nerve coursed dorsal to the ischiopubis and innervated both most of the extensor muscles (vastus lateralis, rectus femoris, and vastus intermedius muscles) and the flexor muscles, unrelated to the muscle compartment by the septum. Additionally, the vastus medialis muscle was innervated by the crural nerve (mammalian femoral nerve‐like nerve) that coursed ventral to the ischiopubis. Thus, the extensor quadriceps femoris muscle is innervated by two types of nerves that take different routes to reach the muscles from the plexus. The innervating branches of the three extensor muscles are speculated to have been re‐routed from the dorsal to the ventral side of the ischiopubis and merged into the crural nerve, thereby giving rise to the mammalian femoral nerve in the tetrapod evolutionary process. This nerve re‐routing of the branches to the extensor muscles is presumably attributed to interspecific differences in hindlimb position and septum structure.

Keywords: dual innervation, femoral nerve, intermuscular septa, quadriceps femoris, sciatic nerve

1. Introduction

The evolutionary trend in the thigh muscles of tetrapods is to increase in number and complexity to meet the demands of locomotion on land and in water. (Kent and Carr 2001). The massive thigh muscles are generally composed of extensor, flexor, and adductor muscles; this anatomical organization is widely observed across mammals. Among mammals, in the human thigh, the lateral and medial femoral intermuscular septa separate these muscle groups, compartmentalizing extensor muscles from flexor/adductor muscles in the posterolateral and posteromedial thigh, respectively (Piersol 1919; Schaeffer 1942).

Among these septal structures, the lateral femoral intermuscular septum arises from the posterior part of the iliotibial tract (part of the fascia lata) as a tendinous lamina (Flato et al. 2017; Gray 1918). The lateral femoral intermuscular septum is located in the posterior lateral thigh and separates the vastus lateralis muscle from the biceps femoris muscle. It is almost vertically fixed to the linea aspera (lateral lip) of the femur by its deep end. The vastus lateralis muscle is innervated by the femoral nerve in anterolateral to the lateral intermuscular septum, and the biceps femoris muscle by the common peroneal and tibial nerves; these two nerves form the sciatic nerve, in posteromedial to the septum (Piersol 1919; Schaeffer 1942). To reach the extensor muscles compartmentalized by the medial and lateral intermuscular septa from the adductor and flexor muscles, the femoral nerve descends ventral to the pubis. The sciatic nerve descends dorsal to the ischium and supplies innervating branches to the flexor muscles in the thigh. From such anatomical characteristics, it is considered that the lateral intermuscular septum not only distinguishes the thigh muscle location but also simultaneously separates the muscular innervation areas by the femoral and sciatic nerves.

In addition to the femoral and sciatic nerves, the obturator nerve is one of the main branches innervating the hindlimb in mammals (Chauveau 1873; Gray 1918; Hartman and Straus Jr 1969). The obturator nerve passes through the obturator foramen (ventral to the ischium and dorsal to the pubis) and innervates the adductor muscles compartmentalized by the medial intermuscular septum from the extensor muscles. Thus, thigh extensor, flexor, and adductor compartments are innervated by nerves that follow different routes: ventral to the pubis (femoral nerve), ventral to the ischium and dorsal to the pubis (obturator nerve), or dorsal to the ischium (sciatic nerve). However, in some amphibians such as frogs, pelvic structures intricately change in conjunction with function (Leavey et al. 2023), and the obturator foramen and nerve are absent (Bolk et al. 1938; Ecker 1889; Sutherland and Nunnemacher 1974). In such amphibian species, the innervation of the thigh muscles and its anatomical relationship to the structural compartmentalization by the intermuscular septa is of morphological interest.

Given this background, we carefully dissected bullfrogs ( Lithobates catesbeianus , Anura) to examine the morphology of thigh muscles and their innervation, with particular attention to the compartmentalization of the thigh muscles. In this article, we focus on the thigh extensor and flexor muscles and their innervations, for which we obtained notable findings and insights. We then discuss these findings from morphological and morphogenetic viewpoints.

2. Materials and Methods

2.1. Specimens

In this study, ten thighs from five bullfrog specimens were dissected and examined macroscopically and partially histologically. These specimens are from the collection of the Department of Physical Therapy, Faculty of Health and Medical Care, Saitama Medical University; they had previously been used for undergraduate physiology experiments. To keep the bullfrog specimens suitable for careful dissection, observations, and data collection (mainly photographs), they were embalmed with 10% formaldehyde and stored in 50% ethanol. The snout‐vent length of the frogs was approximately 180–200 mm. This study was conducted in accordance with the Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan), revised in 2006. No frog was euthanized for the purpose of this study.

2.2. Dissection

The skin around the abdominal, dorsal lumbar, and hindlimb regions was removed. As the superficial fascia closely adhered to the deep surface of the skin and could not be isolated from each other, it was removed with the skin. Some spaces were observed between the superficial fascia (closely layered with skin) and the muscles covered with thin deep fascia in the abdominal, lumbar back, and hindlimb regions; these were termed the subcutaneous lymph sacs (Kent and Carr 2001). When removing the set of skin and superficial fascia from the frog thigh, the intermuscular septum was left intact in the thigh by appropriately cutting the connection between the superficial fascia, deep fascia covering the thigh muscles, and intermuscular septum. After in situ observations of thigh muscles, each muscle was appropriately isolated to observe the attachment site and whole structure of the muscle from multiple angles for accurate identification based on previous reports (Anetai, Kato, et al. 2025; Kimura et al. 2021).

The thoracoabdominal wall was incised and widely opened, and the thoracoabdominal viscera was completely extracted to observe the spinal nerve roots, lumbosacral plexus, and its branches. The muscles of the back and dorsal abdominal wall were partially removed to observe the lumbosacral plexus and its branches from the dorsal aspect. The muscles, intermuscular septum, and nerves observed in the bullfrog pelvis and thigh were individually identified. We additionally performed histological analysis to visualize the lateral intermuscular septum. Embalmed specimens from the dorsal thigh region, including the septum and adjacent muscles, were dehydrated, embedded in paraffin, and sectioned at 8 μm using a conventional sliding microtome. The sections were then stained with haematoxylin and eosin according to a standard protocol. The homology of the identified structures was considered by referring to the literature on amphibian and comparative anatomy (Diogo and Ziermann 2014; Ecker 1889; Leavey et al. 2024; Menzies and Parker 2018; Molnar and Diogo 2021; Přikryl et al. 2009) and detailed human anatomy (Gray 1918; Piersol 1919; Schaeffer 1942). Furthermore, because human anatomy has been studied extensively and many insights into muscle derivation have emerged from analyses of muscle morphology and innervation, we re‐evaluated the thigh muscles in bullfrogs in light of these findings. All structures examined in this study were recorded using photographs and line drawings.

3. Results

Here, we focused on the morphology of the extensor and flexor muscles, their innervations, and lateral intermuscular septum (thigh muscle compartment) in the bullfrog thigh region. The thigh muscles were identified and named on the basis of their proximal and distal attachment sites, location (positional relation to the adjacent muscles and bones), innervations, and combinations of these observations. The observed morphological characteristics of all muscles, as well as the innervation and compartment patterns, were consistent in all specimens.

3.1. Anatomy of Extensors and Flexors in Thighs

Two massive muscular heads were recognized as the extensor muscles, one located on the cranial side of the femur and the other craniodorsal to the femur (Figure 1a). When detaching their proximal origins, we identified that the head on the cranial side originated from the ventral and cranial edges of the acetabulum and hip joint capsule (red area in Figure 1c), whereas the other head on the dorsal side originated from the caudal end of the ilium and tuberosity dorsal to the acetabulum (orange area in Figure 1c), without attachments to the femur. On the basis of their positional relation to the femur and attachment sites, the head on the cranial side is termed the rectus femoris muscle and the other the vastus lateralis muscle. The muscle corresponding to the mammalian vastus medialis muscle was newly identified in this study as a thin and long muscle located on the ventral side of the rectus femoris muscle and deep to the sartorius muscle, partially adhering to the adductor magnus muscle at their distal parts (Figure 1b,b′). The vastus medialis muscle arose from the cranial edge of the pubis (yellow area in Figure 1c).

FIGURE 1.

FIGURE 1

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Thigh extensor muscles in the right bullfrog thigh This figure exhibits the representative case of the extensor muscles in the bullfrog thigh. The dorsal aspect of bullfrog thigh muscles is shown in (a) and the ventral aspect in (b and b'). In (b'), the proximal attachments of the sartorius muscle are detached and distally inverted to exhibit the vastus medialis muscle. The proximal muscular attachment sites of the extensor muscles are indicated on the lateral side of the right pelvis in (c). (d) and (e) show the superficial and deep surfaces of the isolated quadriceps femoris muscle, respectively. The single asterisk indicates the innervating branch to the vastus medialis muscle, and double asterisks indicate the branch to the vastus lateralis, rectus femoris, and vastus intermedius muscles. (f) and (f') show the schematic diagrams of the quadriceps femoris muscle (cranial view). AM, adductor magnus muscle; BF, biceps femoris muscle; Gc, gastrocnemius muscle; RF, rectus femoris muscle; RIMj, rectus internus major muscle; Sa, sartorius muscle; St, semitendinosus muscle; TA, tibialis anterior muscle; VI, vastus intermedius muscle; VL, vastus lateralis muscle; VM, vastus medialis muscle.

In the deep aspect of the rectus femoris muscle, partially divided muscle fascicles from the rectus femoris muscle were observed, which are homologous to the vastus intermedius muscle (Figure 1d,e). These muscular heads of thigh extensor muscles immediately converged and finally attached to the tibial tuberosity and crural fascia with a stout distal tendon (Figure 1d–f,f′).

In the caudal thigh region, thigh flexor muscles were observed (Figures 1a, 2a,a′,b). The dorsal flexor muscle originated from the caudal edge of the tuberosity dorsal to the acetabulum (green area in Figure 2c); at the distal end, it inserted into the proximal region of the tibia. This muscle was located caudal to the lateral intermuscular septum. Based on these findings, it corresponds to the biceps femoris muscle. In addition to the biceps femoris muscle, a small muscle originating from the tuberosity dorsal to the acetabulum was observed proximal and deep to the biceps femoris muscle. It terminated at the proximal femur (green dagger in Figure 2b). This muscle corresponds to the ischiofemoralis muscle based on its attachments and location.

FIGURE 2.

FIGURE 2

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Thigh flexor muscles in the right bullfrog thigh This figure exhibits the representative case of the flexor muscles in the bullfrog thigh. (a) and (a') show the in situ flexor muscles (hamstrings). The white arrowheads indicate the semitendinosus muscle raphe‐like structure. In (a'), the proximal attachments of the rectus internus major and minor muscles are detached and distally inverted to exhibit the semimembranosus and adductor magnus muscles. The two parts of the biceps femoris muscle, the innervating branch of the three heads of the quadriceps femoris muscle passing between the two parts of the biceps femoris (indicated by red arrowheads), semimembranosus, adductor magnus muscles, and partial fusion of the latter two muscles are shown in (b). They become visible by detaching the origin of the semitendinosus and inverting it distally. The green asterisks and green dagger indicate the superficial and deep heads of the biceps femoris muscle, respectively. The proximal muscular attachment sites of the flexor muscles are indicated in the lateral side of the right pelvis in (c). AM, adductor magnus muscle; BF, biceps femoris muscle; Gc, gastrocnemius muscle; RIMj, rectus internus major muscle; RIMn, rectus internus minor muscle; Sm, semimembranosus muscle; St, semitendinosus muscle; VL, vastus lateralis muscle.

A thick and long muscle was located on the superficial layer (Figures 1a, 2a,a′), and this muscle had the semitendinosus muscle raphe‐like structure (white arrowheads in Figure 2a). Based on these findings, it was identified as the semitendinosus muscle in this study. Additionally, a double‐headed muscle was identified at a layer relatively deeper than the semitendinosus muscle and caudal to the adductor magnus muscle (Figure 2a′,b), which was identified as the semimembranosus muscle. Literature on frogs has described the superficial one as the semimembranosus muscle and the relatively deep one as the semitendinosus muscle (Diogo and Ziermann 2014; Ecker 1889; Leavey et al. 2024). However, in contrast to previous descriptions, we interpreted the homologies of these muscles differently, on the basis of the observation, as in many mammals, the semitendinosus muscle is located in the superficial layer of thigh flexor muscles, the semitendinosus muscle raphe‐like structure is observed, and the semimembranosus muscle is in close contact with the adductor magnus muscle (Gray 1918; Nickel et al. 1986; Tojima et al. 2022). The proximal end of the semitendinosus muscle adhered to the ischial tuberosity, with the dorsal head of the semimembranosus muscle (Figure 2c). The ventral head originated from the small prominent area ventral and caudal to the acetabulum and originated with the adductor magnus muscle, and the ventral and dorsal heads fused at their distal ends (Figure 2b). Slender proximal tendons were observed in each head of the semimembranosus muscle. These flexor muscles extended toward the popliteal fossa and finally inserted into the ventral area of the proximal tibia. The rectus internus major and minor muscles in bullfrogs also covered the semimembranosus muscle (Figure 2a,a′).

3.2. Structure of the Lateral Intermuscular Septum

The lateral intermuscular septum continued from the deep surface of the dorsal part of the superficial fascia lata, and the whole fascia lata closely adhered to the deep surface of the skin, as mentioned in the Materials and Methods section. In the dorsal thigh, the lateral intermuscular septum was observed to enter between the vastus lateralis and biceps femoris muscles and anchored to the caudal surface of the femoral shaft (Figure 3a,a′,c). Proximal to the lateral intermuscular septum, the frog gluteal muscles were thin and small and structurally discontinuous to the lateral intermuscular septum. The proximal end of the lateral intermuscular septum was located at the level of the proximal one‐third to one‐fourth of the thigh and the distal end was at the knee (green arrowheads in Figure 3a,a′), and it was more developed and stronger in the distal portion. In addition, another thin septa‐like fascia was observed on the ventral side of the biceps femoris muscle. The medial femoral intermuscular septum was not recognized.

FIGURE 3.

FIGURE 3

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Lateral femoral intermuscular septum in the bullfrog thigh This figure exhibits the representative case of the lateral femoral intermuscular septum of the bullfrog thigh. (a) and (a') show the cranial (extensor side) and caudal (flexor side) surfaces of the lateral femoral intermuscular septum, respectively. The green asterisks indicate the proximal end of the lateral intermuscular septum. (b) shows the histological view of approximate distal one‐third of the lateral intermuscular septum. The yellow arrowheads indicate the lateral intermuscular septum. BF, biceps femoris muscle; Gc, gastrocnemius muscle; LFIS, lateral femoral intermuscular septum; RF, rectus femoris muscle; St, semitendinosus muscle; TA, tibialis anterior muscle; VL, vastus lateralis muscle.

3.3. The Hindlimb Plexus, Its Branches, and Innervations

The lumbosacral plexus originated from the ventral rami of the seventh to tenth spinal nerves (SN7–SN10) and branched out into some branches within the abdominal cavity (Figure 4a,b). Among the branches, the sciatic nerve comprised SN8 and SN9 and ran caudally to emerge from the pelvic cavity passing dorsal to the ischiopubis. After the sciatic nerve appeared in the deep layer of the gluteal region, it supplied muscular branches to the thigh flexors and notably to the vastus lateralis, rectus femoris, and vastus intermedius muscles (Figure 4a–c). The branch distributing to the three thigh extensor muscles arose from SN8 and SN9 as a part of the common peroneal nerve division of the sciatic nerve (Figures 4a,b, 5a,b,b′) and ran caudally, passing between the ischiofemoralis and biceps femoris muscles (red arrowheads in Figure 2b). Subsequently, it coursed more proximal than the proximal end of the lateral intermuscular septum and divided to the branches reaching the vastus lateralis, rectus femoris, and vastus intermedius muscles (Figure 4b,c).

FIGURE 4.

FIGURE 4

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The lumbosacral plexus and its branches in the right half of the pelvis of the bullfrog This figure shows the representative case of the right lumbosacral plexus and its branches in the right half pelvis of the bullfrog. The dorsal view of the lumbosacral plexus and its branches is shown by photograph in (a) and by drawing in (b). To show the plexus and its branches from the dorsal aspect, the back and dorsal abdominal wall muscles are removed. The green arrowheads indicate the innervating branch to the vastus lateralis, rectus femoris, and vastus intermedius muscles. (c) shows the ramification and termination of this innervating branch. The green asterisk indicates the main trunk of the muscular branch; the yellow, red, and orange arrowheads indicate the branch to the vastus lateralis, rectus femoris, and vastus intermedius muscles, respectively. The ventral view of the lumbosacral plexus and its branches is shown by photograph in (d) and by drawing in (e). The orange arrowheads indicate the muscular nerve to the vastus medialis muscle. BF, biceps femoris muscle; CP, common peroneal nerve; Cr, crural nerve; LFIS, lateral femoral intermuscular septum; Pe, pectineus muscle; Pu, pudendal nerve; RF, rectus femoris muscle; SA, sciatic artery; ScN, sciatic nerve; Ti, tibial nerve; VI, vastus intermedius muscle; VL, vastus lateralis muscle; VM, vastus medialis muscle.

FIGURE 5.

FIGURE 5

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Origin of the innervating branch to the three muscular heads of the right quadriceps femoris muscle This figure shows the representative case of the origin of the innervating branch to the vastus lateralis, rectus femoris, and vastus intermedius muscles. The nerve fibres that compose this innervating branch are traced proximally by removing loose connective tissues around the nerve to identify which nerve root this branch originates from. The sciatic nerve is also divided into the tibial and common peroneal nerve divisions using the same procedure. The tibial and common peroneal nerve divisions of the sciatic nerve and the muscular branch to the three heads of the quadriceps femoris muscle are shown by photograph in (a) and by drawing in (b) and (b'). The green asterisks indicate the muscular branch to the three heads of the quadriceps femoris muscle. BF, biceps femoris muscle; CP, common peroneal nerve; RF, rectus femoris muscle; St, semitendinosus muscle; Ti, tibial nerve; VI, vastus intermedius muscle; VL, vastus lateralis muscle.

The vastus medialis muscle was innervated by the thin nerve from SN7 and SN8 (orange arrowheads in Figure 4d,e). It arose from the bifurcation of the common trunk formed by itself and innervating branch of the pectineus muscle and ran ventral to the ischiopubis. This common trunk is termed the crural nerve in frog anatomy and had some characteristics similar to those of the mammalian femoral nerve (see Discussion section). Therefore, the quadriceps femoris muscle was innervated by two types of hindlimb main nerves, which take different routes to reach the muscles from the plexus.

4. Discussion

In this study, we carefully examined the extensor and flexor muscles, their innervation by the hindlimb plexus branches, and their relationship to the lateral intermuscular septum (muscle compartment) in bullfrog thighs. Through the examination, we characterized these structures and found that the bullfrog quadriceps femoris muscle is innervated by two types of nerves: the crural nerve (a mammalian femoral nerve‐like nerve) and a common peroneal division of the sciatic nerve that is not related to the thigh muscle compartments. This pattern clearly differs from that in mammals. In the following section, we discuss these findings from the viewpoints of morphology and morphogenesis.

4.1. Muscle Identification

In this study, thigh muscles in bullfrogs were primarily identified based on classical and modern anatomy textbooks and studies on amphibians. Additionally, consideration of human anatomy, in which knowledge has been overwhelmingly accumulated compared to that of other species, contributed to the new identification of muscles such as the vastus medialis, semitendinosus and semimembranosus muscles (Table 1). In comparative anatomy, considering muscle innervation in addition to muscle structure has provided helpful insights into muscle identification, including considerations of homology and muscle derivation (Anetai et al. 2026; Emura et al. 2020, 2023; Koizumi 2019, 2022, 2023; Sakuraya et al. 2023; Velez‐Garcia and Miglino 2023), which has also been partially corroborated by experimental embryological studies using chick embryos (Nagashima et al. 2020). Nerve fibre analysis by peeling the epineuria performed in some of the abovementioned studies would be useful and reliable.

TABLE 1.

Muscle nomenclature used in current and previous literature.

Present study Ecker (1889) Marshall (1920) Přikryl et al. (2009) Diogo and Ziermann (2014) Leavey et al. (2024)
Extensors

Quadriceps femoris

 Rectus femoris Triceps femoris Rectus anterior Cruralis Cruralis Cruralis
Vastus lateralis Vastus externus Gluteus maximus Extensor iliotibialis B (gluteus maximus) Gluteus maximus
Vastus intermedius Vastus internus Not mentioned Not mentioned Not mentioned
Vastus medialis Not mentioned Adductor longus Not mentioned Not mentioned
Sartorius Sartorius Sartorius Pubotibialis A (sartorius) Sartorius
Flexors Biceps femoris Iliofibularis (biceps) Iliofibularis Tenuissimus (iliofibularis) Iliofibularis
Ischiofemoralis Not mentioned Ischiofemoralis Not mentioned Not mentioned
Semitendinosus Semimembranosus Semimembranosus Ischioflexorius (semimembranosus) Semimembranosus
Semimembranosus Ventral head Semitendinosus Anterior head Semitendinosus Pubotibialis B (semitendinosus) Semitendinosus Ventral head
Dorsal head Posterior head Dorsal head
Adductors (not all) Pectineus Pectineus Pectineus Pubioischiofemoralis A (pectineus) Pectineus
Rectus internus major Rectus internus major Gracilis major and minor Gracilis major Gracilis major and minor
Rectus internus minor Rectus internus minor Gracilis minor
Adductor magnus Adductor magnus Adductor magnus Adductor femoris (adductor magnus) Adductor magnus
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However, we should be cautious in applying anatomical descriptions of mammalian muscles and their innervations, including those of humans, to species with widely divergent phylogenies such as amphibians in this case when identifying their muscles. Several discrepancies were observed between the traditional nomenclature and the actual morphological characteristics in this study, such as the biceps femoris, semitendinosus, and semimembranosus muscles. Koizumi (2023) examined shoulder girdle muscles in domestic fowl and newly classified them on the basis of their innervation. In this manner, flexibility in identifying and characterizing them according to the morphological characteristics of each species without being restricted by conventional descriptions would be important.

4.2. Anatomy of the Hindlimb Plexus and the Innervating Branches to the Quadriceps Femoris Muscle

In bullfrogs, the lumbosacral plexus comprised SN7–SN10 and its branches innervated the hindlimb. The crural nerve arose from SN7 and SN8, coursed ventral to the pubis, and finally innervated the vastus medialis muscle of the quadriceps femoris muscle and pectineus muscle. This nerve is considered homologous to the femoral nerve owing to its relation to the branch of the pectineus muscle, coursing (positional relation to the pubis), and distribution similar to that of the mammalian femoral nerve. Aizawa (1992) previously reported that, in humans, the innervating branch to the vastus medialis muscle forms a common trunk with the branch to the pectineus muscle, which also corroborates our considerations. The remaining vastus lateralis, rectus femoris, and vastus intermedius muscles were innervated by the nerve arising from SN8 and SN9, included in the proximal part of the common peroneal nerve division of the sciatic nerve, and it coursed dorsal to the ischiopubis to terminate in the muscles.

According to previous literature on amphibians and reptiles, the lumbosacral plexus and its branches have not been explained in much detail. Only Ecker (1889) and Brown et al. (1989) briefly mentioned a nerve extending toward the thigh extensor muscles from the sciatic nerve, although they did not specifically describe or indicate this nerve in any of their diagrams or photographs. In addition, Ecker (1889) appeared to illustrate this nerve as a thigh and/or knee sensory nerve in one of the figures.

4.3. Morphogenetic Hypothesis: Nerve Re‐Routing Within the Hindlimb Plexus in the Tetrapod Evolutionary Process

In this study, the origin, branching pattern, and dual innervation (femoral nerve and common peroneal nerve) of the quadriceps femoris muscle were clearly characterized and described in detail as typical patterns. Cruce (1974) reported that lumbosacral plexus roots occasionally showed intraspecific variation of the cranial or caudal deviation in frogs, which is also similarly observed in the mammalian lumbosacral plexus and its branches (Anetai et al. 2022; Anetai, Teramoto, et al. 2025; Ishiguro et al. 2020; Tokita et al. 2021; Tubbs et al. 2018) and may be related to variations in nerve routing (Akita et al. 1994). In this study, remarkable variations including the two branches to the quadriceps femoris muscle were not identified. These two nerves originate from adjacent roots but pass ventral or dorsal to the ischiopubis to reach each head of the quadriceps femoris muscle. Thus, although the origin and termination of the two nerves are close, their routes differ notably. In mammals, nerves arising from common roots (lumbosacral plexus roots) and innervating the thigh extensor, flexor, and adductor compartments course through different routes as follows: ventral to the pubis (femoral nerve), ventral to the ischium and dorsal to the pubis (obturator nerve), or dorsal to the ischium (sciatic nerve including common peroneal and tibial divisions) (Chauveau 1873; Gray 1918; Hartman and Straus Jr 1969; Paterson 1887; Raven 1950; Sepulveda‐Vasquez and Tamayo‐Arango 2023). The innervating branches of the three heads of the quadriceps femoris muscle are speculated to have been re‐routed from the dorsal to the ventral side of the ischiopubis and merged into the femoral nerve, thereby giving rise to the mammalian femoral nerve in the tetrapod evolution. This re‐routing of the branches to the extensor muscles is presumably attributed to interspecies differences in hindlimb position and lateral intermuscular septum structure.

Based on illustrations in various animal literature, the bullfrog hip joint (thigh) is usually in maximum abduction, whereas the mammal hip joint is generally more adducted than that of bullfrogs. In mammals, the quadriceps femoris muscle is therefore positioned ventral to the pubis, as illustrated by Nickel et al. (1986), and it is structurally reasonable for the innervating branches of the quadriceps femoris muscle to course ventral to the pubis to reach the muscles. Furthermore, in primates including humans, the lateral intermuscular septum develops from proximal to distal as part of the fascia lata to provide the muscular attachment sites for gluteal and thigh muscles (Anetai, Kato, et al. 2025; Raven 1950; Schaeffer 1942; Sisson and Grossman 1943; Sparteholz 1939). Such lateral intermuscular septum and adjacent muscles possibly structurally obstruct the extending of the innervating branch of thigh extensor muscles from dorsal to ventral (the migration of the precursor undifferentiated cells of the nerve in the early fetal period) in the lateral thigh in primates.

However, the radial and ulnar nerves perforate the brachial intermuscular septa from dorsal to ventral in humans (Piersol 1919; Schaeffer 1942), and this may differ between species (Raven 1950). Therefore, the relationship of nerves travelling between the ventral and dorsal regions to the intermuscular septa should be further considered in terms of comparative anatomy and morphogenesis. For example, what are the interspecific and/or intraspecific differences among regions (e.g., between fore‐ and hindlimbs, and among the thigh and lower leg regions) in the relationship of the nerve to septa? Additionally, what are the embryological and developmental processes of the nerves, septa, and adjacent muscles, in terms of heterochronic differences in the timing and rate of their development? At present, detailed anatomical descriptions of muscle innervation and topographical relationships to adjacent structures are inadequate for addressing this topic in comparative anatomy; therefore, it would be beneficial to accumulate such information in future studies.

Literature has reported several human anatomic variations that appear to be related to nerve re‐routing. The accessory obturator nerve is well‐known case. This atypical nerve descends ventral to the pubis and reaches the proximal adductors and hip joint capsule (Hollinshead 1982; Tubbs et al. 2018; Turgut et al. 2017), which is interpreted as a case of partial re‐routing of the obturator nerve to the ventral side. Akita et al. (1994) reported that the superior gluteal nerve innervated the rectus femoris and vastus lateralis muscles in humans; this case is well similar to quadriceps femoris muscle innervation in bullfrogs revealed in our study. They described that the muscular branch of the rectus femoris and vastus lateralis muscles may travel via the superior gluteal nerve and is related to the cranial deviation of the lumbosacral plexus and its branches. From the perspective of comparative anatomy, Anetai et al. (2023) examined the brachial plexus and its branches in several primates and suggested that the cutaneous nerve distributing to the posteromedial aspect of the upper arm changes its origin and route corresponding to interspecies differences in the shoulder joint range of motion. In experimental embryology, Homma et al. (2022) interpreted anomalous rectus abdominis muscle innervation (dual innervation by typical and extra nerves) as resulting from nerve fibres that, instead of changing their route to merge into the typical nerve, remain as an extra nerve. This interpretation was based on their original three‐component model of the spinal nerve, which they proposed on the basis of their embryological examination of the intercostal nerve in mouse embryos. Such findings including the present results that seem to be related to nerve re‐routing are expected to be helpful in understanding the morphogenesis of anatomic variations, interspecific diversity in muscle innervations, and the origin of dual innervation. Additionally, in arterial embryology, the relationship between femoral artery development and sciatic artery regression in the fetal period is known as representative arterial re‐routing (Arey 1956; Senior 1919). Although a difference exists between whether it occurs during ontogenesis or phylogenesis, if dynamic nerve re‐routing occurs as in the artery, as we suggested, it would provide new implications for comparative embryology and evolutionary morphology.

4.4. Limitation and Perspectives

In this study, we particularly focused on the extensor and flexor muscles, their innervations, and intermuscular septa in bullfrog thighs. The morphological characterizations and considerations of the septum and lumbosacral plexus branches were primarily conducted with reference to descriptions of human anatomy, due to a lack of relevant information in other species. Further similar exploration in other species—such as non‐human primates, non‐primate mammals, reptiles, and amphibians—would be helpful to critically evaluate, refine, and expand our present findings and suggestions.

In this article, the adductor muscles and their innervations were only briefly and concisely mentioned. We are currently investigating them in bullfrogs and plan to report our findings in detail and thoroughly discuss them in a future article, as the absence of the obturator foramen and the obturator nerve and their relation to the adductor muscles are distinct characteristics in frogs and present a complicated issue. Identifying their anatomical characteristics would be an important step toward understanding thigh muscle innervation and nerve re‐routing during tetrapod evolution.

5. Conclusions

This study clearly characterized and described in detail the anatomy of the bullfrog quadriceps femoris muscle and its distinct innervation, including adjacent structures such as the lateral intermuscular septum and flexor muscles. The crural (femoral) and common peroneal nerve divisions of the sciatic nerves innervate the quadriceps femoris muscle; specifically, the vastus medialis muscle by the crural (femoral) nerve and the vastus lateralis, rectus femoris, and vastus intermedius muscles by the nerve from the common peroneal nerve division, unrelated to the muscle compartment (by the lateral intermuscular septum). The innervating branches of the three heads of the quadriceps femoris muscle are speculated to have been re‐routed from the dorsal to the ventral side of the ischiopubis and merged into the crural (femoral) nerve, thereby giving rise to the mammalian femoral nerve during tetrapod evolution. This nerve re‐routing is presumably attributed to interspecies differences in hindlimb position and in the lateral intermuscular septum structure. These findings are helpful for understanding the morphogenesis of anatomic variations and interspecific diversity in muscle innervations and the origin of dual innervation.

Author Contributions

H.A. and K.T. conceptualization, methodology, data curation, validation, writing – original draft, writing – review and editing, resource, supervision. M.W. and M.O. preliminary examinations. S.A. resource, writing – review and editing. T.A. and R.K. methodology, writing – review and editing.

Funding

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgements

We are also grateful to Dr. Ryosuke Goto (Gunma Paz University) for his insightful comments. Finally, we thank Enago (www.enago.jp) for English language editing.

Contributor Information

Hidaka Anetai, Email: h-anetai@juntendo.ac.jp.

Kounosuke Tokita, Email: tokita@saitama-med.ac.jp.

Data Availability Statement

On reasonable request, the data obtained in this study are available from the corresponding author.

References

  1. Aizawa, Y. 1992. “On the Organization of the Plexus Lumbalis. I. On the Recognition of the Three‐Layered Divisions and the Systematic Description of the Branches of the Human Femoral Nerve.” Okajimas Folia Anatomica Japonica 69, no. 1: 35–74. [DOI] [PubMed] [Google Scholar]
  2. Akita, K. , Sakamoto H., and Sato T.. 1994. “A Case in Which a Branch From the Superior Gluteal Nerve Innervated the Rectus Femoris and the Vastus Lateralis.” Annals of Anatomy 176: 181–183. [DOI] [PubMed] [Google Scholar]
  3. Anetai, H. , Kato K., Kudoh H., Sakai T., and Ichimura K.. 2025. “Structural Re‐Evaluation of the Human Gluteus Maximus.” Scientific Reports 15, no. 1: 21251. 10.1038/s41598-025-05361-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Anetai, H. , Teramoto J., Ono T., et al. 2025. “Twelfth Rib Length as a Predictor of Anatomical Variations in the Lumbosacral Plexus Associated With Atypical Radiculopathy in Lumbar Disc Herniation.” Spine 50: E505–E512. 10.1097/BRS.0000000000005400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Anetai, H. , Tokita K., Kojima R., Hirasaki E., Sakai T., and Ichimura K.. 2022. “Relationship Between the Lumbosacral Plexus Deviation and 12th Rib Length in Japanese Macaques ( Macaca fuscata ).” Anatomical Science Intetnational 97, no. 4: 391–398. 10.1007/s12565-022-00657-8. [DOI] [PubMed] [Google Scholar]
  6. Anetai, S. , Tokita K., Anetai H., et al. 2023. “Morphological Significance of the Medial Brachial Cutaneous Nerve: An Anatomical Study of the Brachial Plexus in Primates.” Primates 64, no. 2: 261–272. 10.1007/s10329-022-01046-5. [DOI] [PubMed] [Google Scholar]
  7. Anetai, S. , Tokita K., Anetai H., Kojima R., Hirasaki E., and Endo H.. 2026. “Comparative Anatomy of the Levator Scapulae, Serratus Anterior and Rhomboid Muscles in Primates: Morphological Adaptations Related to the Primate Locomotion Evolution.” Journal of Anatomy 249: 247–263. 10.1111/joa.70100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Arey, L. B. 1956. Developmental Anatomy. W. B. Saunders. [Google Scholar]
  9. Bolk, L. , Göppert E., Kallius E., and Lubosch W.. 1938. Handbuch der vergleicheden Anatomie der Wirbeltiere. Urban & Schwarzenberg. [Google Scholar]
  10. Brown, D. R. , Everett A. W., and Bennett M. R.. 1989. “Compartmental and Topographical Distributions of Axons in Nerves to the Amphibian ( Bufo marinus ) Glutaeus Muscle.” Journal of Comparative Neurology 284, no. 2: 231–241. 10.1002/cne.902840207. [DOI] [PubMed] [Google Scholar]
  11. Chauveau, A. 1873. The Coparative Anatomy of the Domesticated Animals. D. Appleton & Co. [Google Scholar]
  12. Cruce, W. L. 1974. “The Anatomical Organization of Hindlimb Motoneurons in the Lumbar Spinal Cord of the Frog, Rana Catesbiana.” Journal of Comparative Neurology 153, no. 1: 59–76. 10.1002/cne.901530106. [DOI] [PubMed] [Google Scholar]
  13. Diogo, R. , and Ziermann J. M.. 2014. “Development of Fore‐and Hindlimb Muscles in Frogs: Morphogenesis, Homeotic Transformations, Digit Reduction, and the Forelimb–Hindlimb Enigma.” Journal of Experimental Zoology Part B: Molecular and Developmental Evolution 322, no. 2: 86–105. [DOI] [PubMed] [Google Scholar]
  14. Ecker, A. 1889. The Anatomy of the Frog. Clarendon Press. [Google Scholar]
  15. Emura, K. , Hirasaki E., and Arakawa T.. 2020. “Muscle‐Tendon Arrangement and Innervation Pattern of the m. Flexor Digitorum Superficialis in the Common Marmoset ( Callithrix jacchus ), Squirrel Monkey ( Saimiri sciureus ) and Spider Monkey (Ateles sp.).” Journal of Anatomy 237, no. 5: 907–915. 10.1111/joa.13250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Emura, K. , Hirasaki E., and Arakawa T.. 2023. “Muscle‐Tendon Arrangement and Intramuscular Nerve Distribution of Flexor Digitorum Superficialis in the Siamang ( Symphalangus syndactylus ), Western Lowland Gorilla ( Gorilla gorilla ), Western Chimpanzee ( Pan troglodytes verus ), and Japanese Macaque ( Macaca fuscata ).” Anatomical Science International 98, no. 4: 493–505. 10.1007/s12565-023-00713-x. [DOI] [PubMed] [Google Scholar]
  17. Flato, R. , Passanante G. J., Skalski M. R., Patel D. B., White E. A., and Matcuk G. R. Jr. 2017. “The Iliotibial Tract: Imaging, Anatomy, Injuries, and Other Pathology.” Skeletal Radiology 46, no. 5: 605–622. 10.1007/s00256-017-2604-y. [DOI] [PubMed] [Google Scholar]
  18. Gray, H. 1918. Anatomy of the Human Body. 20th ed. Lea & Febiger. [Google Scholar]
  19. Hartman, C. G. , and Straus W. L. Jr. 1969. The Anatomy of the Rhesus Monkey. Hafner Publishing Co. [Google Scholar]
  20. Hollinshead, W. H. 1982. “Anatomy for Surgeons.” In Vol. 3: The Back and Limb, Lippincott Williams & Wilkins. [Google Scholar]
  21. Homma, S. , Shimada T., Wada I., Kumaki K., Sato N., and Yaginuma H.. 2022. “A Three‐Component Model of the Spinal Nerve Ramification: Bringing Together the Human Gross Anatomy and Modern Embryology.” Frontiers in Neuroscience 16: 1009542. 10.3389/fnins.2022.1009542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ishiguro, K. , Kawashima T., and Sato F.. 2020. “The Phenotypic Morphology of Human Lumbar Plexus Roots Associated With Changes in the Thoracolumbar Vertebral Count and Trade‐Off.” Scientific Reports 10, no. 1: 127. 10.1038/s41598-019-56709-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kent, G. C. , and Carr R. K.. 2001. Compative Anatomy of the Vertebrates. McGraw‐Hill. [Google Scholar]
  24. Kimura, N. , Kato K., Anetai H., et al. 2021. “Anatomical Study of the Soleus: Application to Improved Imaging Diagnoses.” Clinical Anatomy 34, no. 7: 991–1001. 10.1002/ca.23667. [DOI] [PubMed] [Google Scholar]
  25. Koizumi, M. 2019. “Two Mammalian Species in Which the Intercostal Nerves Innervate the Serratus Anterior or Scalenus Muscles Together With the Cervical Nerves: An Important Clue to Clarify the Homology of Cervico‐Thoracic Trunk Muscles in Mammals.” Anatomical Science International 94, no. 4: 295–306. 10.1007/s12565-019-00487-1. [DOI] [PubMed] [Google Scholar]
  26. Koizumi, M. 2022. “Comparative Anatomy of the Subscapularis, Teres Major and Latissimus Dorsi Muscles From Salamanders to Mammals With Special Reference to Their Innervations From the Brachial Plexus.” Anatomical Science International 97, no. 1: 124–137. 10.1007/s12565-021-00636-5. [DOI] [PubMed] [Google Scholar]
  27. Koizumi, M. 2023. “A New Classification of the Shoulder Girdle Muscles in Domestic Fowl Based on Their Innervation From the Brachial Plexus.” Anatomical Science International 99, no. 2: 202–214. 10.1007/s12565-023-00751-5. [DOI] [PubMed] [Google Scholar]
  28. Leavey, A. , Richards C. T., and Porro L. B.. 2024. “Comparative Muscle Anatomy of the Anuran Pelvis and Hindlimb in Relation to Locomotor Mode.” Journal of Anatomy 245, no. 5: 751–774. 10.1111/joa.14122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Leavey, A. , Ruta M., Richards C. T., and Porro L. B.. 2023. “Locomotor, Ecological and Phylogenetic Drivers of Skeletal Proportions in Frogs.” Journal of Anatomy 243, no. 3: 404–420. 10.1111/joa.13886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Marshall, A. M. 1920. The Frog: An Introduction to Anatomy, Histology, and Embryology. Macmillan. [Google Scholar]
  31. Menzies, J. , and Parker F.. 2018. “The Natural History and Head and Shoulder Anatomy of Cornufer guentheri (Anura, Ceratobatrachidae), a Casque‐Headed Frog of the Solomon Islands.” Alytes 35, no. 1–4: 17–38. [Google Scholar]
  32. Molnar, J. L. , and Diogo R.. 2021. “Evolution, Homology, and Development of Tetrapod Limb Muscles.” Diversity 13, no. 8: 393. 10.3390/d13080393. [DOI] [Google Scholar]
  33. Nagashima, H. , Koga D., Kusumi S., et al. 2020. “Novel Concept for the Epaxial/Hypaxial Boundary Based on Neuronal Development.” Journal of Anatomy 237, no. 3: 427–438. 10.1111/joa.13219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nickel, R. , Schunmer A., Seiferle E., Frewein J., Wilkens H., and Wille K. H.. 1986. The Anatomy of the Domestic Animals. Vol. 1. Verlag Paul Parey. [Google Scholar]
  35. Paterson, A. M. 1887. “The Limb Plexuses of Mammals.” Journal of Anatomy and Physiology 21, no. 4: 611–634. [PMC free article] [PubMed] [Google Scholar]
  36. Piersol, G. 1919. Human Anatomy. 7th ed. J. B. Lippincott & Co. [Google Scholar]
  37. Přikryl, T. , Aerts P., Havelková P., Herrel A., and Roček Z.. 2009. “Pelvic and Thigh Musculature in Frogs (Anura) and Origin of Anuran Jumping Locomotion.” Journal of Anatomy 214, no. 1: 100–139. 10.1111/j.1469-7580.2008.01006.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Raven, H. C. 1950. The Anatomy of the Gorilla. Columbia University Press. [Google Scholar]
  39. Sakuraya, T. , Sekiya S. I., Emura K., Sonomura T., Hirasaki E., and Arakawa T.. 2023. “Comparison of the Soleus and Plantaris Muscles in Humans and Other Primates: Macroscopic Neuromuscular Anatomy and Evolutionary Significance.” Anatomical Record (Hoboken) 306, no. 2: 386–400. 10.1002/ar.24999. [DOI] [PubMed] [Google Scholar]
  40. Schaeffer, J. 1942. Morris' Human Anatomy: A Complete Systematic Treatise. 10th ed. Blakiston Company. [Google Scholar]
  41. Senior, H. 1919. “The Development of the Arteries of the Human Lower Extremity.” American Journal of Anatomy 25, no. 1: 54–95. [Google Scholar]
  42. Sepulveda‐Vasquez, A. , and Tamayo‐Arango L.. 2023. “Lumbar Vertebral Pattern Variation in the Common Opossum ( Didelphis marsupialis Linnaeus, 1758): Implication on Lumbar Nerve Distribution.” International Journal of Veternary Science and Medicine 11, no. 1: 1–10. 10.1080/23144599.2022.2163561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sisson, S. , and Grossman J. D.. 1943. The Anatomy of the Domestic Animals. W. B. Saunders Co. [Google Scholar]
  44. Sparteholz, W. 1939. Handatlas der Anatomie des Menschen. Verlag von S. Hirzel. [Google Scholar]
  45. Sutherland, R. M. , and Nunnemacher R. F.. 1974. “Fibers in the Ventral Spinal Nerves of the Frog.” Journal of Comparative Neurology 156, no. 1: 39–47. 10.1002/cne.901560105. [DOI] [PubMed] [Google Scholar]
  46. Tojima, S. , Anetai H., Koike K., et al. 2022. “Gross Anatomy of the Gluteal and Posterior Thigh Muscles in Koalas Based on Their Innervations.” PLoS One 17, no. 9: e0261805. 10.1371/journal.pone.0261805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tokita, K. , Anetai H., Kojima R., et al. 2021. “Relationship of Segmental Variations in the Human Lumbar Plexus to the Length of the 12th Rib.” Annals of Anatomy 233: 151592. 10.1016/j.aanat.2020.151592. [DOI] [PubMed] [Google Scholar]
  48. Tubbs, R. S. , Loukas M., Hanna A. S., and Oskouian R. J.. 2018. Surgical Anatomy of Lumbar Plexus. Thieme Medical Pub. [Google Scholar]
  49. Turgut, M. , Protas M., Gardner B., Oskouian R. J., Loukas M., and Tubbs R. S.. 2017. “The Accessory Obturator Nerve: An Anatomical Study With Literature Analysis.” Anatomy 11, no. 3: 121–127. 10.2399/ANA.17.043. [DOI] [Google Scholar]
  50. Velez‐Garcia, J. F. , and Miglino M. A.. 2023. “Evolutionary Comparative Analysis of the Extrinsic Thoracic Limb Muscles in Three Procyonids ( Procyon cancrivorus Cuvier, 1798, Nasua Linnaeus, 1766, and Potos flavus Schreber, 1774) Based on Their Attachments and Innervation.” Anatomical Science International 98, no. 2: 273–292. 10.1007/s12565-022-00696-1. [DOI] [PubMed] [Google Scholar]

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Data Availability Statement

On reasonable request, the data obtained in this study are available from the corresponding author.

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