Morphological features of the orbicularis oculi muscle and facial nerve in four odontocete families, with comparisons within Cetartiodactyla

Abstract

We examined the orbicularis oculi (OC) muscle and its innervation among the markedly modified facial muscles of odontocetes, clarifying features related to secondary adaptations to aquatic life. The OC muscle has been described as a well-developed sphincter encircling the eye in cetaceans; however, this study revealed two distinct types of OC morphology among odontocetes. In specimens from the families Kogiidae and Ziphiidae, the muscle bundles of the OC completely encircled the palpebral fissure in concentric circles, similar to that of Bovidae specimens. However, in specimens from Phocoenidae and Delphinidae, the OC featured a muscular area with muscle fibers and an aponeurotic area comprising collagen fibers lacking facial nerve distribution. Our results suggest that this aponeurotic area was acquired in the common ancestor of species within the Delphinoidea superfamily, and that the upper eyelid is less mobile than the lower eyelid. The palpebral branches, which originate from the facial nerve, extend from the lower to the upper palpebral regions in the examined odontocetes, showing a clear difference from the entry course seen at the lateral canthus in artiodactyls. The proximal palpebral branches diverged directly from the facial nerve trunk, forming a characteristic diverging point distal to that of artiodactyls. These branches probably correspond to the auriculopalpebral nerve, which has lost the rostral auricular branch component. The morphological changes in cetacean heads also led to changes in the branching pattern and pathway of the facial nerve.

INTRODUCTION

The facial muscles around the nostril of cetaceans have undergone significant changes related to the modification of cranial bone arrangement, known as telescoping, especially in odontocetes. The development of echolocation organs and the loss of the auricle evolved as part of adapting to the aquatic environment. As a result, many studies on odontocete facial muscles have focused on structures involved in echolocation surrounding the blowhole, including the melon and air sacs [6, 8, 13, 16, 20]. In contrast, the orbicularis oculi (OC) muscle, which is one of the facial muscles and encircles the palpebral fissure with muscle fibers, has received little attention. Its anatomical studies have only described a well-developed sphincter muscle surrounding the eye, which seems to have been observed incidentally in research primarily focused on nasal muscles around the blowhole and auricular, mandibular, or ocular muscles [12, 17, 22]. However, the eyes of cetaceans also have features adapted to the underwater environment [11, 18, 26], such as well-developed Harderian glands and retractor bulbi muscles [9], with extraocular muscles that are more specialized than those of other mammals [22]. Therefore, the OC muscle, which functions to close the eyelid, is also likely adapted to underwater conditions compared to terrestrial mammals.

Additionally, although limited, some studies have described the facial nerve of odontocetes [5, 10, 12, 28]. Overall, these studies have shown that the facial nerve, after exiting the stylomastoid foramen, forms a nerve trunk, runs rostrally under the zygomatic bone until the antorbital notch (formed laterally in the maxilla and lacrimal at the base of the rostrum [21]), and then turns to the surface of the skull roof, dividing into branches that innervate the blowhole musculature. Only Huber [12] and Schulte and Smith [28] described the branches innervating the OC muscle that branch off beneath the orbit, while Cozzi et al. [5] documented that the facial nerves of dolphins run without further branching from the stylomastoid foramen along the zygomatic arch to the antorbital notch. In terrestrial mammals, the main terminal branches of the facial nerve typically diverge in the proximal region after exiting the stylomastoid foramen [3, 4]. The branching pattern of the proximal facial nerve in odontocetes is poorly understood, and the intramuscular distribution of the facial nerve remains unknown, requiring clarification for comparison with terrestrial mammals. Based on molecular and morphological studies, the Cetacea have been confirmed to belong to the Artiodactyla/Cetartiodactyla clade, with Hippopotamidae as the sister group to the Cetacea [2, 7, 19, 23, 24, 33]. Comparing cetaceans and artiodactyls allows us to consider the drastic morphological evolutionary changes that have occurred in cetaceans. The OC muscle among facial muscles appears to be more similar between odontocetes and artiodactyls than the specialized nasal muscles or rudimentary auricular muscles found in odontocetes adapted to aquatic life, and is a suitable part for comparison. We aimed to shed light on the homologous and evolutionarily specific features involved in aquatic adaptation, focusing on the OC muscle and its innervation branch, by comparing odontocetes and artiodactyls within the same clade. Updating the anatomical description of the OC muscle and its innervation in odontocetes will hopefully contribute to our understanding of the radical changes in facial structure that occurred during the evolution from terrestrial to aquatic habitats.

MATERIALS AND METHODS

Dissection

We examined the morphology of 48 OC muscles and 12 facial nerves from 36 odontocete heads representing 15 species collected at the National Museum of Nature and Science (Tokyo, Japan; Table 1). All individuals died from stranding, incidental catch, or were kept in an aquarium, with specimen numbers biased by species. A bharal (Pseudois nayaur) carcass from a zoo, belonging to the family Bovidae, was also examined for comparison within the same clade [7] (Table 1). Dissections were performed on raw or 10% formalin-fixed samples using a scalpel, tweezers, and a Zeiss OPMI 111 surgical stereomicroscope (Carl Zeiss Microscopy GmbH, Jena, Germany) as necessary. Soft facial tissues, including the OC muscle, facial nerve, and ocular contents, together with blubber, were removed en bloc from the skull and fixed in 10% formalin in most cases. For observation after formalin fixation, the ocular contents and blubber were removed from the head and fixed in 10% formalin. The fat and connective tissues were carefully removed under the surgical stereomicroscope to expose the surface of the OC muscle and the facial nerve. The findings were recorded photographically (using a Nikon D850 camera) and through line drawings by tracing the photographs.

Table 1. Specimens used in the study.

NMNS ID number	Infraorder	Family	Species	Common name	Body length (cm)	Sex	Side	Method applied
M57764	Cetacea	Kogiidae	Kogia sima	Dwarf sperm whale	232.5	M	R	Dissected OC muscle and facial nerve, Sihler’s staining
M62797	Cetacea	Ziphiidae	Mesoplodon ginkgodens	Ginkgo-toothed beaked whale	480	M	L, R	Dissected OC muscle
M65325	Cetacea		Mesoplodon ginkgodens	Ginkgo-toothed beaked whale	ca. 519	F	L, R	Dissected OC muscle and facial nerve
M65332	Cetacea		Mesoplodon stejnegeri	Stejneger’s beaked whale	499.2	F	L, R	Dissected OC muscle and facial nerve
M65363	Cetacea		Mesoplodon stejnegeri	Stejneger’s beaked whale	476.3	F	R	Dissected OC muscle
M65400	Cetacea		Mesoplodon stejnegeri	Stejneger’s beaked whale	485	F	L	Dissected OC muscle and facial nerve
M84704	Cetacea		Mesoplodon stejnegeri	Stejneger’s beaked whale	344	F	L, R	Dissected OC muscle
M88303	Cetacea		Ziphius cavirostris	Cuvier’s beaked whale	518.7	F	L, R	Dissected OC muscle
M84800	Cetacea		Berardius minimus	Sato’s beaked whale	689	M	R	Dissected OC muscle
M54906	Cetacea	Phocoenidae	Phocoenoides dalli	Dall’s porpoise	ca. 200	M	R	Dissected OC muscle
M72171	Cetacea		Phocoenoides dalli	Dall’s porpoise	196.5	M	R	Dissected OC muscle and facial nerve
M54854	Cetacea		Neophocaena asiaeorientalis	Finless porpoise	120.4	F	R	Dissected OC muscle
M54855	Cetacea		Neophocaena asiaeorientalis	Finless porpoise	116.9	M	L, R	DissectedOC muscle and facial nerve, L: Sihler’s staining, R: histological sections
M57844	Cetacea		Neophocaena asiaeorientalis	Finless porpoise	74.4	M	R	Dissected OC muscle
M55014	Cetacea	Delphinidae	Stenella coeruleoalba	Striped dolphin	235	F	L, R	Dissected OC muscle
M55016	Cetacea		Stenella coeruleoalba	Striped dolphin	229.2	M	L, R	Dissected OC muscle and facial nerve, LR: Sihler’s staining
M55018	Cetacea		Stenella coeruleoalba	Striped dolphin	220.5	F	L, R	Dissected OC muscle, R: histological sections
M62790	Cetacea		Stenella coeruleoalba	Striped dolphin	227.5	M	L, R	Dissected OC muscle
M65306	Cetacea		Stenella coeruleoalba	Striped dolphin	218.8	F	L, R	Dissected OC muscle and facial nerve, R: Sihler’s staining
M72257	Cetacea		Stenella coeruleoalba	Striped dolphin	233.4	M	L	Dissected OC muscle
M72258	Cetacea		Stenella coeruleoalba	Striped dolphin	202.2	F	R	Dissected OC muscle and facial nerve
M72261	Cetacea		Stenella coeruleoalba	Striped dolphin	218.3	F	R	Dissected facial nerve
M65356	Cetacea		Grampus griseus	Risso’s dolphin	238.5	M	L	Dissected OC muscle
M65357	Cetacea		Grampus griseus	Risso’s dolphin	249.1	M	L	Dissected OC muscle
M72181	Cetacea		Grampus griseus	Risso’s dolphin	180	F	R	Dissected OC muscle
M84694	Cetacea		Grampus griseus	Risso’s dolphin	218.3	M	L, R	Dissected OC muscle
M72330	Cetacea		Peponocephala electra	Melon-headed whale	247	F	L	Dissected OC muscle
M72331	Cetacea		Peponocephala electra	Melon-headed whale	255	F	L	Dissected OC muscle
M72332	Cetacea		Peponocephala electra	Melon-headed whale	236	F	L, R	Dissected OC muscle
M72333	Cetacea		Peponocephala electra	Melon-headed whale	232.7	M	L	Dissected OC muscle
M72335	Cetacea		Peponocephala electra	Melon-headed whale	249.7	F	R	Dissected OC muscle and facial nerve
M62795	Cetacea		Globicephala macrorhynchus	Short-finned pilot whale	350.5	F	L	Dissected OC muscle
M65382	Cetacea		Lagenodelphis hosei	Fraser’s dolphin	230	M	R	Dissected OC muscle
M84678	Cetacea		Steno bredanensis	Rough-toothed dolphin	232.6	F	L	Dissected OC muscle
M84680	Cetacea		Tursiops truncactus	Bottlenose dolphin	261	F	L	Dissected OC muscle
M84801	Cetacea		Aethalodelphis obliquidens	Pacific white-sided dolphin	210	F	R	Dissected OC muscle
M80227	Pecora	Bovidae	Pseudois nayaur	Bharal	119	M	R	Dissected OC muscle and facial nerve

NMNS: National Museum of Nature and Science, OC: orbicularis oculi muscle.

The terminology used in this study, specifically regarding cetaceans, was adopted from publications by Huber [12], Mead and Fordyce [21], and Meshida et al. [22]. For other terminologies, we used those from Budras et al. [3, 4].

Histological sections

Horizontally sectioned muscle samples were obtained from the upper and lower palpebral regions of one striped dolphin (Stenella coeruleoalba, M55018, R) and one finless porpoise (Neophocaena asiaeorientalis, M54855, R). These samples were embedded in paraffin at the National Museum of Nature and Science. The slides were sectioned into 4 μm slices and stained using hematoxylin and eosin (HE), Masson’s trichrome (MT) to identify collagen fibers, and Elastica van Gieson (EVG) stains for elastic fibers at the Hissan Medical Support Center Co. (Utsunomiya, Japan), following standard protocols. Samples were also collected from the medial and lateral canthus regions to examine the muscle fiber connections between the upper and lower eyelids. All slides were examined under a Zeiss Axio Imager M1 light microscope (Carl Zeiss Microscopy GmbH). Photographs were taken using a Zeiss AxioCam HRc, ZEN 3.4 Blue Pro Software, and a Nikon D800 camera.

Sihler’s staining

The soft tissues, including the OC muscle and facial nerve, were fixed in 10% formalin and stained using Sihler’s technique, following the method described by Sekiya et al. [29], to examine the intramuscular distribution of the facial nerve branches. Sihler’s stain is a whole-mount nerve staining technique that renders other soft tissues translucent or transparent with glycerin, while staining nerves a deep blue-purple with Ehrlich’s hematoxylin. Samples of one dwarf sperm whale (Kogia sima, M57764, R), one finless porpoise (M54855, L), and two striped dolphins (M55016, L, R; M65306, R) were used. After staining, the facial nerve was observed from the medial aspect using a Zeiss OPMI 111 surgical stereomicroscope (Carl Zeiss Microscopy GmbH). The intramuscular nerve distribution was visualized by removing the soft tissue, except for the blue-stained nerves. The sketches were prepared based on photographs.

RESULTS

Dissection of the OC muscle

The OC muscle was attached to the orbital rim, and the outer ventral fibers attached to the auriculolabial (zygomatic) muscle or fat across all examined odontocete specimens. Figure 1 shows a representative example from each of the four odontocete families. The muscle fascicles of the OC muscle completely encircled the palpebral fissure in the dwarf sperm whale (Fig. 1A and 1A’). In all four species of ziphiids examined, the OC muscle resembled that of the dwarf sperm whale; however, horizontal muscle fascicles were present on its surface (Fig. 1B and 1B’). These horizontal muscle fascicles originated from the pre- and post-orbital processes of the frontal bone [21] and the zygomatic process of the squamosal bone, and inserted into the blubber near the lateral and medial canthi. Some fibers of the dorsolateral rim of the OC muscle attached to the post- and pre-orbital processes. Conversely, in the two Dall’s porpoises (Phocoenoides dalli) and three finless porpoises examined, the muscle fascicles did not completely encircle the eye opening as an entirely muscular structure; a part of the upper palpebral region was degenerated to form an aponeurotic area (Fig. 1C and 1C’). These OC muscles comprised an aponeurotic area made of tendinous tissue and a muscular area. The tendinous tissues appeared to be intermediate tendons connecting the muscular fibers on both sides. The aponeurotic area was observed not only in the upper palpebral region but also in the medial canthus region in all eight delphinid species examined (Fig. 1D and 1D’). The tendinous tissues in the medial canthus region appeared to be the tendons of origin for the muscular fibers of the upper and lower palpebral regions, as these tissues arose from the preorbital process and connected to the muscle fibers of the upper and lower eyelids.

Fig. 1.

The orbicularis oculi (OC) muscle of odontocetes in the lateral view. The left row of the figure shows photographs taken from the right (A–C) and the left (D) side of the head, with their respective illustrations shown in the right row. The skin and blubber have been removed. (A, A’) The muscle fascicles of the OC surround the entire eye opening in Kogia sima (M57764). (B, B’) The surface of the OC, composed entirely of muscle bundles, contains horizontal muscle fascicles (asterisk) that originate from the Pep and Pop and insert into the blubber near the lateral and medial canthi of Mesoplodon stejnegeri (M65363). (C, C’) Neophocaena asiaeorientalis (M54855). The muscle fascicles of OC do not entirely surround the eye opening. The upper palpebral region contains an aponeurotic area (dashed line area) of tendinous tissue. (D, D’) The OC contains aponeurotic areas in the upper palpebral and medial canthus region of Globicephala macrorhynchus (M62795). AL, auriculolabial muscle; D, dorsal; E, ear; Pep, preorbital process; Pop, postorbital process; R, rostral.

On the other hand, in a bharal of the family Bovidae, the muscle fascicles of the OC muscle encircled the orbital rim and palpebral fissure (Fig. 2). The muscle fascicles were thinner and the tint was lighter than those of odontocetes. It was also identified that the retractor anguli oculi lateralis muscle is located at the caudal rim of the OC muscle.

Fig. 2.

The right facial muscles and the facial nerve (yellow), artery (magenta), and vein (skyblue) around the eye in the lateral view of the examined bharal (Pseudois nayaur, M80227). A is an enlarged photograph of the white box in the right corner of the image. A’ is a sketch based on A. Arrowheads indicate the nerve branch supplying the orbicularis oculi (OC) muscle. The branch enters from the lateral canthus and diverges from the auriculopalpebral nerve (APN). D, dorsal; fro, frontalis muscle; fs, frontoscutular muscle; mal, malaris muscle; mdp/msp, deep/superficial part of masseter muscle; R, rostral; ral, retractor anguli oculi lateralis muscle; STA/V, superficial temporal artery/vein; za, zygomaticoauricular muscle.

Dissection of the facial nerve

The facial nerve trunk of all examined odontocetes, emerging from the stylomastoid foramen, ran under the zygomatic bone and surface of the masseter, giving off several small branches. All branches expected to innervate the OC muscle entered the auriculolabial (zygomatic) muscle beneath the OC muscle. In a dwarf sperm whale (Fig. 3A and 3A’), after giving off the small branches heading for the external auditory meatus, a nerve about one-third the thickness of the facial nerve trunk (Fig. 3A”, asterisk) diverged dorsally at an angle of approximately forty-five degrees between the posterior end of the palpebral fissure and the external auditory meatus. Among the smaller branches that further diverged from this branch, some directed toward the OC muscle, while others joined the facial nerve trunk or communicated with distal branches diverging from the trunk toward the OC muscle. Additionally, just before giving off that one-third-thickness nerve, three nerves formed the entire trunk with repeated branching and anastomosing rather than as a single bundle (Fig. 3A”); the bundling in the facial nerve appeared weaker than in the specimens from the other three families. Conversely, the branching pattern of the nerve that appeared to innervate the OC muscle was similar among the three ziphiid, two phocoenid, and five delphinid specimens that were dissected (Fig. 3B–D). The most proximal branches heading toward the OC muscle diverged directly from the dorsal side of the facial nerve trunk, positioned between the anterior tip of the squamosal bone (squamous part of the temporal) and the caudal tip of the palpebral fissure, at nearly right angles. This diverging point (Fig. 3B”–D”, solid star) was a distinct characteristic compared to other distal branches. In the specimens from ziphiids (Fig. 3B’ and 3B”) and delphinids (Fig. 3D’ and 3D”), the branches that diverged from this characteristic point were the thickest, while the branches in the phocoenid specimens (Fig. 3C’ and 3C”) did not differ in thickness from the distal branches and diverged radially. Among the nerves diverging from this point, some connected to the nerve trunk or distal branch, while others extended to the OC muscle. Besides, just before that characteristic diverging point, the facial nerve trunk was enveloped in dense tendinous tissue in these specimens, forming a highly bundled single trunk within the tissue (Fig. 3B”–D”), which differed from the kogiid specimen.

Fig. 3.

The facial nerve trunk below the eye and its nerve branches (yellow), arteries (magenta), and veins (skyblue) of the examined odontocetes in the medial view. A’–D’ are drawn from A–D, respectively. A, B, and D are on the right side, while C is on the left. A”–D” shows enlarged sketches corresponding to the black rectangles in each image of A’–D’. The eyeball and ocular muscles have been removed in A, while in others, the eyeball and ocular muscles, including the external circular muscle (ECM), remained. Arrowheads indicate the nerve branches expected to innervate the orbicularis oculi (OC) muscle, which diverge from the dorsal side of the facial nerve trunk. (A, A’, A”) Kogia sima (M57764). Asterisk indicates a nerve branch that diverged from the facial nerve trunk, including the most proximal branch directed to the OC muscle. (B, B’, B”) Mesoplodon ginkgodens (M65325). (C, C’, C”) Neophocaena asiaeorientalis (M54855). (D, D’, D”) Stenella coeruleoalba (M72261). At the solid star, branches diverge directly from the facial nerve trunk, and this branch is thicker than the other distal branches, with a characteristic diverging point compared to the others. Scale bars: 2 cm, D, dorsal; E, ear; FV, facial vein; MA, maxillary artery; R, rostral; II, optic nerve; VII, facial nerve.

In the bharal, the OC muscle was innervated by the palpebral branch, which diverged from the zygomatic branch of the auriculopalpebral nerve (Fig. 2A’). The auriculopalpebral nerve coursed dorsally between the auricle and the zygomatic arch, diverging into the rostral auricular branch and the zygomatic branch. The palpebral branch passed beneath the retractor anguli oculi lateralis muscle and entered the OC muscle from the lateral angle.

Histological sections

One striped dolphin (Fig. 4A–C and 4A’–C’) and a finless porpoise revealed that the aponeurotic area of the upper palpebral region comprised collagen and elastic fibers. The muscle fibers were discontinuous in the deep layers of the aponeurotic area. In only the striped dolphin, three sections were collected from the upper eyelid, and the elastic fibers were more abundant near the palpebral fissure than around the rim of the OC muscle (Fig. 4A”–C”). In contrast, the lower palpebral region was composed solely of muscle fibers (Fig. 4D and 4D’). Thus, the aponeurotic area, which was observed grossly, was confirmed histologically, and the OC muscle fascicles of phocoenids and delphinids were not completely encircled.

Fig. 4.

The horizontal cross section of the right orbicularis oculi (OC) muscle in a striped dolphin (Stenella coeruleoalba, M55018) specimen. Sections stained with hematoxylin and eosin (A–D), Masson’s trichrome (A’–D’), and Elastica van Gieson (A”–C”) were taken from the same samples. (A–C, A’–C’) The section from the upper palpebral region of the OC muscle. The muscle fibers (mf) of the OC muscle within the aponeurotic area are not continuous and are disrupted. The area consists of collagen fibers (cf). (A”–C”) Corresponds to the black rectangle in the A’–C’ image. The enlarged image corresponds to the white rectangle in each image. The elastic fibers (ef, arrowheads) are more abundant near the palpebral fissure. (D) The section from the lower palpebral region of the OC muscle. The muscle fibers of the OC muscle are continuous in this section. (E) The positions from which each section sample was taken. Lps, levator palpebrae superioris muscle.

Figure 5 shows the histological images of OC muscle tissue obtained from the medial and lateral canthus regions to observe the boundary between the upper and lower palpebral regions. In the case of the finless porpoise, the outer muscle fibers were interconnected in the upper and lower eyelids (Fig. 5A–B). Some horizontal muscle fibers were found in the medial canthus region (Fig. 5B’), whereas they were clearly observed in the lateral canthus region (Fig. 5A’). In the medial canthus region of striped dolphins, the aponeurotic area did not contain the muscle fibers, and the muscle fibers extended to the upper and lower palpebral regions from the aponeurotic area (Fig. 5D’). The horizontal muscle fibers were identified within the superficial layer of the OC muscle, situated between the aponeurotic area in the medial canthus region and the medial palpebral fissure, with an increased number of fibers observed approximately one millimeter deeper. Conversely, only a few horizontal muscle fibers were found in the lateral canthus region, and the outer muscle fibers were interconnected in the upper and lower regions (Fig. 5C’). The current histological analysis could not ascertain the origin of the horizontal muscle fibers because the samples were collected after removing the OC muscle and orbital content.

Fig. 5.

The hematoxylin and eosin-stained surface layers of the right orbicularis oculi muscle in the lateral (A, C) and medial (B, D) canthus, with illustrations showing the muscle fiber direction (A’–D’) in the lateral view. (A, B) Neophocoena asiaeorientalis (M54855). (C, D) Stenella coeruleoalba (M55018). The muscle fibers are observed to traverse horizontally within the red squares, in contrast to those in the upper and lower palpebral regions. The aponeurotic area (Apo) in the medial canthus region (D, D’) lacks muscle fibers. D, dorsal; R, rostral.

Sihler’s staining

Figure 6 shows representative examples of the intramuscular distribution of the facial nerve in the dwarf sperm whale, where the OC muscle consists entirely of muscle fibers, and in the striped dolphin, which features an aponeurotic area. Based on Sihler’s stain specimen observations, the branches innervating the OC muscle passed the auriculolabial (zygomatic) muscle, entered from the ventral side, and distributed throughout the lower palpebral region in all examined specimens. Many thin branches diverging from the small branches were distributed in the upper palpebral region of the dwarf sperm whale, mainly on the caudal side (Fig. 6A’). A few small branches diverged and reached the upper palpebral region in the striped dolphin (Fig. 6B’) and the finless porpoise; however, they were not distributed in the aponeurotic area. Some branches, diverging from the characteristic point, reached the lower and upper palpebral regions, while others communicated with the distal branches of the facial nerve or the branches expected to be the ophthalmic nerve. There were no branches that directed to the external auditory meatus from the characteristic diverging point. In addition to the muscular branches of the OC muscle, many small branches arose from the ventral and dorsal aspects of the facial nerve trunk, though their distribution was not identified. Additionally, a few communicating branches with the mandibular nerve (V3) were also observed in one striped dolphin at a more rostral location than the OC muscle and ventral to the facial nerve trunk (Fig. 6B’). In a Kogiidae specimen, we observed communication between the facial nerve branch and a branch that comes from profundus to superficial at the same location and along the same nerve course as that of the striped dolphin (Fig. 6A’). We could not identify the origin of the branch; however, it was conceivable that it was the mandibular nerve.

Fig. 6.

The photographs (A, B) and sketches (A’, B’) of the facial nerve (VII, yellow) intramuscular distribution of the right OC muscle based on Sihler’s stained specimen in the medial view. After removing the tendinous tissue surrounding the facial nerve trunk, both were drawn based on the photograph. All nerve branches to the orbicularis oculi (OC) muscle extend from the lower to the upper palpebral regions. The nerve, which seemed to be the ophthalmic nerve (V1, gray), passed through the OC and reached the superficial skin layer. (A, A’) Kogia sima (M57764). The nerve distribution around the ear is not shown here. (B, B’) Stenella coeruleoalba (M65306). There is no facial nerve distribution in the aponeurotic area. The solid star indicates the characteristic diverging point of the branches innervating the OC. D, dorsal; E, ear; R, rostral; V3, mandibular nerve.

DISCUSSION

Morphological features of the OC muscle in cetaceans

In this study, we revealed that the OC muscle in odontocetes has two types of morphologies depending on whether or not it has an aponeurotic area in the upper eyelid region (Fig. 7a). The OC muscles of terrestrial mammals, including the bharal examined, as well as those of mysticetes, such as the bowhead whale (Balaena mysticetus) [34] and the humpback whale (Megaptera novaeangliae) [25], are entirely composed of muscular fibers. Thus, among odontocetes, the OC muscles of the Kogiidae and Ziphiidae families retained their typical mammalian or ancestral forms. The species in the Kogiidae and Ziphiidae families are known as deep-diving species [1, 32]. The short-finned pilot whale (Globicephala macrorhynchus), a member of the Delphinidae family, is also considered a deep diver [30], but we found that it has an aponeurotic area in its OC muscle. The presence of the aponeurotic area in the OC muscle is likely a genetic characteristic rather than an acquired characteristic, as it has been identified only in the phylogenetically recent families of Phocoenidae and Delphinidae within the odontocetes. Huber [12] described the OC muscle of the bottlenose dolphin (Tursiops truncatus) and the narwhal (Monodon monoceros) but did not mention the presence of the aponeurotic area. Although we did not examine any Monodontidae species, it is conceivable that they may have an aponeurotic area in the OC muscle because they are considered closely related to Phocoenidae species and belong to the Delphinoidea superfamily (Fig. 7a), which is the clade uniting Delphinidae, Phocoenidae, and Monodontidae [7, 19]. Therefore, we suggest that the common ancestor of Delphinidae, Phocoenidae, and Monodontidae acquired the aponeurotic area in the OC muscle. Further research is needed to clarify why only phocoenid and delphinid species possess an aponeurotic area in the OC muscle. At the very least, the vestibular sac related to echolocation, found only in members of the Delphinoidea and Iniidae species, is considered a derived character; on the other hand, ziphiids lack it [8]. It is possible that species in the Delphinoidea superfamily developed the aponeurotic area as part of their head modifications, including echolocation-related organs for aquatic life.

Fig. 7.

Morphological diagram of the orbicularis oculi (OC) muscle and the facial nerve distribution on the skull and mandible. Each illustration shows the left side. a) The relationship between OC muscle morphology and the simplified phylogenetic tree. The phylogenetic tree was referenced and revised from Hassainin et al. [7]. The OC muscle in Mysticeti and Monodontidae was cited from Rodrigues et al. [25], Zhu et al. [34], and Huber [11]. The OC muscle consists of muscle fibers overall in Boviidae, Kogiidae, and Ziphiidae specimens, while the muscular area and the aponeurotic area, primarily composed of collagen fibers, are also present in Phocoenidae and Delphinidae specimens. The horizontal muscle fascicles on the surface of the OC muscle in Ziphiidae likely correspond to the levator anguli oculi medialis and retractor anguli oculi lateralis muscles in terrestrial mammals. b–f) The following species are shown as representatives of their respective families: Pseudois nayaur, Kogia sima, Mesoplodon ginkgodens, Neophocaena asiaeorientalis, and Stenella coeruleoalba. The skull and mandible were modified to the angle for the condylobasal axis (dashed line, CBL). The solid star indicates the diverging point of the auriculopalpebral nerve (palpebral branch) innervating the OC muscle. The orbital position of odontocetes, shown by the OC muscle, is located more ventrally than that of bovids, descending to the condylobasal axis line. The diverging point of the palpebral branch (conceivably corresponding to the auriculopalpebral nerve) of odontocetes is located more distally than that of bovids, including the bharal, and the course to the OC muscle differs between odontocetes and artiodactyls.

In terrestrial mammals, including artiodactyls, the medial palpebral ligament secures the medial canthus, while the retractor anguli oculi lateralis muscle pulls the lateral canthus caudally. The horizontal muscle fascicles observed on the surface of the OC muscle in ziphiid specimens probably correspond to the levator anguli oculi medialis and retractor anguli oculi lateralis muscles in terrestrial mammals, as described in the bowhead whale [34]. The aponeurotic area in the medial canthus region of delphinids may contribute to maintaining the palpebral fissure position during OC muscle contraction, similar to the function of the medial palpebral ligament. Histological analysis of Phocoenidae and Delphinidae specimens revealed that muscle fibers run horizontally at the medial and lateral canthi. Therefore, these muscular fibers pull on the canthi, and the OC muscle is thought to close the upper and lower eyelids using the medial and lateral canthi as fulcrums. Because the horizontal pulling structure of the palpebral fissure could not be observed grossly in Kogiidae, further detailed investigation of this structure will be necessary in the future.

Comparison of the facial nerve branching pattern of the OC muscle

The diagram of the course and distribution of the facial nerve examined in the present study is shown in Fig. 7b–f. In bovines, the auriculopalpebral nerve (N. auriculopalpebralis), one branch of the facial nerve, supplies the OC and rostral auricular muscles [3]. This innervation pattern is typical in most terrestrial mammals, as confirmed by the bharal specimen in this study (Fig. 7b). However, in cetaceans, they had lost the auricle and scutiform cartilage, and the auricular muscles had degenerated, remaining only as rudimentary muscles surrounding the external auditory meatus [12]. The facial nerve examined in this study was observed as a great bundled nerve trunk that ran under the eye and reached the periphery of the blowhole, as described by Schulte and Smith [28] and Huber [12]. The auriculopalpebral nerve, as found in artiodactyls, was not identified because no nerve branches that distributed to both the OC muscle and the rudimentary muscles surrounding the external auditory meatus were detected. However, many small branches arose from the facial nerve trunk, and the dorsal branches of the trunk distributed to the OC muscle; the most proximal branches formed the characteristic diverging point beneath the eye in the ziphiid, phocoenid, and delphinid specimens (cf. Figs. 3 and 7d–f). Schulte and Smith [28] and Huber [12] did not mention this diverging point; instead, it was observed in a similar position across the examined specimens from each of the three families. The branches arising from the diverging point may correspond to the auriculopalpebral nerve that has lost its rostral auricular branch component. The palpebral branches that innervate the OC muscle in odontocetes, which diverge beneath the zygomatic bone, have shifted their diverging point further distally compared to those in artiodactyls. Additionally, the pathway of the palpebral branch has changed in odontocetes, entering and distributing the OC muscle from the ventral side, while in artiodactyls, it enters from the dorsocaudal side. The acquisition of the palpebral branch pathway entering from the ventral side―unseen in artiodactyls―and its divergence point distally are thought to result from the following factors (Fig. 7b–f): 1) the nostril shifting to the parietal region through telescoping; 2) the ventral descent of the orbital position relative to the condylobasal axis; and 3) the loss of the auricle and degeneration of the auricular muscles. The dorsal buccal branch, which supplies the muscles of the upper lip and nose, ascends dorsally toward the vertex alongside the nostril’s position, as discussed by Huber [12]. Additionally, the auriculopalpebral nerve descended ventrally in line with the orbital position. As the auricle disappeared, the auriculopalpebral nerve lost its rostral auricular branch component. The auriculopalpebral nerve, which lost the rostral auricular branch component, runs close to the dorsal buccal branch and shares the same path until the diverging point of the palpebral branch. The palpebral branch, which diverged near the OC muscle, may have provided a pathway for innervation from the ventral side, resulting in the distally diverging point. Therefore, in odontocetes, the drastic morphological changes in the head during aquatic adaptation have led to modifications in the branching of the facial nerve, resulting in different branching patterns compared to those of artiodactyls.

Huber [12] noted that the branches of the facial nerve came to lie very close together and were consequently enclosed in a heavy connective tissue sheath, with the complex network existing within this sheath. We also confirmed the complex network in the heavy sheath surrounding the facial nerve trunk, but it was only found after the most proximal palpebral branches diverged from a characteristic point in the ziphiid, phocoenid, and delphinid specimens. In contrast, in the examined dwarf sperm whale, nerve branching and anastomosing were observed before the first palpebral branch was given off, and the first palpebral branch originated from the nerve that diverged from the trunk. Since this was not observed in the specimens from the three families, the facial nerve trunk of the kogiid specimen seemed to be incompletely bundled. Schulte and Smith [28] described the facial nerve of the pygmy sperm whale (Kogia breviceps), but did not mention the degree of bundling. The nerve branch shown in Fig. 3A (asterisk) of the kogiid specimen was reminiscent of the state that implies the auriculopalpebral nerve (palpebral branch) descending ventrally due to the previously mentioned modification of head morphology, especially the position of the orbit. The appearance of the difference in the bundling of the facial nerve trunk between the kogiid specimen and those of the other three families (cf. Fig. 3A”–D”) is believed to be related to phylogenetic effects. Of the odontocetes in the present study, Kogiidae species is the most phylogenetically ancient [7, 19], suggesting that the family may be transitioning to a highly bundled facial nerve. However, the morphology of the head in the Kogiidae species is more specialized than that of the groups after the Ziphiidae family, and further examination with an increased number of specimens is required to confirm our findings.

Aponeurotic area of the OC muscle and eyelid movements

The OC muscles of the examined phocoenid and delphinid species contained aponeurotic areas in the upper eyelid and/or medial canthus regions. On slice images of dolphin heads [10, 14] and frontal views of living dolphins, the eyes are positioned in the middle of the head, with the dorsal portion of the dolphin eyeball protruding more than the ventral portion. The aponeurotic area of the upper eyelid corresponded to the most protruding part of the eyeball. Additionally, the ocular structures of cetaceans feature thick sclera and cornea that provide mechanical strength [18] and are remarkably adapted to the underwater environment. Kellogg [15] also described that the eyes of cetaceans have no tarsus or supporting cartilage in the eyelid. Therefore, the upper eyelid of dolphins, corresponding to the most protruding part of the eyelid, may have degenerated into collagen fibers and formed an aponeurotic area as a substitute for the tarsal plate or supporting structure, or to reinforce the eyelid. It is thought that elastic fibers were more abundant near the palpebral fissure than at the rim of the OC muscle due to the OC muscle’s higher extensibility. The differences in OC muscle composition between the upper and lower palpebral regions affect eyelid movement. Based on our findings, the upper eyelid contained the aponeurotic area and had few facial nerve distributions, suggesting that the upper eyelid is likely less mobile than the lower eyelid. The lower eyelids of living dolphins moved more widely and smoothly than their upper eyelids during eyelid movements when we observed these movements. The upper eyelid, composed of muscle and collagen fibers, prevents the extension of opening the eyelid compared to the lower eyelid, which is composed only of muscle fibers. Therefore, the aponeurotic area in the OC muscle is considered to cause the upper eyelid to be less open than the lower eyelid, preventing the delicate movement of the upper eyelid and allowing the lower eyelid to move predominantly. In addition, cetaceans have massive retractor bulbi muscles [9]. When the eyelid closes, the OC muscle contracts to shut the eyelids, and the retractor bulbi muscle also contracts to pull the protruding eyeball back. As a result, the upper eyelid will be considered to fall naturally and assist eyelid closure. The species with an aponeurotic area in the upper eyelids, such as Dall’s porpoise, finless porpoise, striped dolphin, Risso’s dolphin (Grampus griseus), melon-headed whale (Peponocephala electra), short-finned pilot whale, Fraser’s dolphin (Lagenodelphis hosei), rough-toothed dolphin (Steno bredanensis), bottlenose dolphin, and Pacific white-sided dolphin (Aethalodelphis obliquidens), may have a weaker eyelid closure force compared to species in the families Kogiidae and Ziphiidae that have entirely muscle fibers.

In general, blinking in terrestrial mammals helps protect the eyes from drying out and regulates the amount of light entering the eyes. However, because cetaceans possess well-developed Harderian glands [27, 31], blinking seems unnecessary for maintaining the moisture of the corneal surface. Additionally, the pupil shape of some mysticetes and odontocetes, except for the Amazon river dolphin (Inia geoffrensis), is known to be a characteristic U-shaped slit at high illumination, considered a protection from downwelling light rather than upwelling light [18, 26]. Although this pupil shape is not unique to species in the delphinid and phocoenid families, when the collagen fibers in the aponeurotic area prevent the upper eyelid from opening, the upper eyelid may serve to reduce the amount of sunlight entering the eye. In cetaceans with downward-directed visual axes [15], the aponeurotic area of the upper eyelid is considered a structure that contributes to securing the downward sight.

CONFLICTS OF INTEREST

The authors have no conflicts of interest to disclose.