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. 2016 Feb 8;228(5):877–886. doi: 10.1111/joa.12438

Comparative anatomy of the extraocular muscles in four Myliobatoidei rays (Batoidea, Myliobatiformes)

Carlo M Cunha 1,2,✉, Luciano E Oliveira 3, José R Kfoury Jr 4
PMCID: PMC4831342  PMID: 26853799

Abstract

Extraocular muscles are classically grouped as four rectus and two oblique muscles. However, their description and potential associations with species behavior are limited. The objective was to characterize extraocular muscles in four Myliobatoidei rays from diverse habitats with divergent behaviors. Heads (10 per species) of Dasyatis hypostigma, Gymnura altavela, Mobula thurstoni and Pteroplatytrygon violacea were decalcified and dissected to characterize and describe extraocular muscles. Principal component analysis (PCA) was used to evaluate relationships between muscle length and species; for P. violacea, D. hypostigma and G. altavela, these were qualitatively and quantitatively consistent with the general pattern of extraocular muscles in vertebrates. In contrast, for M. thurstoni, the two oblique muscles were completely fused and there was a seventh extraocular muscle, named m. lateral rectus β (both were apparently novel findings in this species). There were also significant differences in eye disposition in the chondrocranium. The PCA axis 1 (rectus muscles) and PCA axis 2 (oblique muscles) accounted for 98.47% of data variability. Extraocular muscles had significant differences in length and important anatomical differences among sampled species that facilitated grouping species according to their life history. In conclusion, extraocular muscles are not uniform in all vertebrate species, thereby providing another basis for comparative studies.

Keywords: Dasyatis, Gymnura, Mobula, morphology, Pteroplatytrygon

Introduction

Elasmobranchs have great morphological diversity, occupy disparate environments and include many species with a highly specialized sensory system, consistent with their roles as top predators in marine ecosystems (Compagno, 1990; Pough et al. 1999; Nelson, 2006). Sharks and rays are generally described as having small eyes and poor vision, relying primarily on other senses (e.g. smell, hearing and electroreception). Nevertheless, after discovery of their double retina containing cones and rods (Gilbert, 1963; Gruber et al. 1963; Hamasaki & Gruber, 1965; Cohen, 1989), more evidence emerged regarding eye function, thereby confirming the importance of vision in several of these species (Gruber & Cohen, 1978; Hueter & Cohen, 1991; Hart et al. 2006).

Elasmobranchs have a variety of advanced visual features, including mobile pupils, multiple visual pigments (essential for color vision), prominent visual streaks (enhance visual acuity) and a moveable lens that facilitate accommodation, characteristics of a much more complex system than previously reported (Hueter, 1991; Hueter et al. 2004). Species with relatively smaller eyes tend to be coastal dwellers (e.g. benthic, batoids and sharks). Furthermore, active benthopelagic and pelagic species that prey on active, mobile prey also have relatively larger eyes than more sluggish benthic elasmobranchs that feed on more sedentary prey such as benthic invertebrates (Lisney & Collin, 2007).

Eyes have functional and structural variations determined by environmental, evolutionary and genetic factors (Hairston et al. 1982). In fish, eye morphology can vary, depending on feeding strategy and habitat, as well as other factors (Menezes et al. 1981; Hairston et al. 1982; Williamson & Keast, 1988; Fanta et al. 1994).

Extraocular muscles are responsible for eyeball movement, and are regarded as constant in number, arrangement and innervation in vertebrates (Goodrich, 1986). There are four rectus and two oblique muscles (Nomina Anatomica Veterinaria, 2007); during eyeball movement, these muscles form three pairs (antagonistic and synergistic). Inferior and superior rectus muscles are responsible for movement around the horizontal axis of the eyeball, thereby directing the eye up or down. Medial and lateral rectus muscles are responsible for movement around the vertical axis (moving eyes forward and backwards). Finally, inferior and superior oblique muscles are responsible for rotation of the eyeball around the optical axis (Harder, 1975; Carpenter, 1977). The innervation pattern by three cranial nerves is consistent among extant vertebrates (Fritzsch et al. 1990). Nerve IV innervates superior oblique. Nerve VI innervates lateral rectus in chondrichthyan and osteichthyan fishes, but two muscles in the lamprey, and in most tetrapods. All remaining extraocular muscles are innervated by Nerve III (Fritzsch et al. 1990; Young, 2008).

Elasmobranchs from different habitats have differences in size of extraocular muscles, perhaps due to reduced use of a particular eye movement, based on habitat and mode of locomotion (Graf and Brunken, 1984). The objective of this study was to characterize extraocular muscles in four Myliobatoidei ray species from diverse habitats with divergent behaviors. The hypothesis that there are significant differences among species in extraocular muscles morphometry was tested. It was expected that results could provide a new basis for future evolutionary/phylogenetic studies and comparative homology within myliobatoids.

Materials and methods

Four species (each from a different genera) were studied within the suborder Myliobatoidei, as proposed by Aschliman et al. (2012) as follows: Dasyatidae: Dasyatis hypostigma; Pteroplatytrygon violacea; Gymnuridae: Gymnura altavela; and Myliobatidae: Mobula thurstoni. Prior to preparation, species were identified according to Figueiredo (1977), Gadig et al. (2003) and Santos & Carvalho (2004). There were 10 individual specimens for each species.

Material consisted mainly of fresh specimens captured by trawling and longline fishing vessels off the coast of São Paulo state, south‐eastern Brazil. Specimens were decalcified and dissected (Casas et al. 2005). Identification and terminology for muscle description followed Nishida (1990) and Shirai (1992). Anatomical descriptions were made with both eyes; however, morphometric analyses were conducted only with right eyes.

All anatomical analyses were conducted at the Faculdade de Medicina Veterinária da Universidade de São Paulo (FMV‐USP). Anatomical abbreviations: CR, chondrocranium; EB, equator of the bulb (central portion of axial length on the ocular bulb); EYL, eye length; Eyt, eyestalk; IR, m. inferior rectus; ITD, insertion tendon; LR, m. lateral rectus; LRβ, m. lateral rectus β; MR, m. medial rectus; IO, m. inferior oblique; OB, ocular bulb; SO, m. superior oblique; SR, m. superior rectus; II, optic nerve (cranial nerve II); III, oculomotor nerve (cranial nerve III); IV, trochlear nerve (cranial nerve IV); VI, abducent nerve (cranial nerve VI). For clarity, photographs of the right eye in Fig. 1 were inverted (horizontally) to match the orientation of muscles in Figs 2, 3, 4 and 6.

Figure 1.

Figure 1

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Right eyeball (flipped horizontally) and extraocular muscles arrangement from Dasyatis hypostigma. (A) Dorsal view of m. superior rectus (SR), m. medial rectus (MR) and mm. oblique (SO and IO) with part of the chondrocranium (CR) attachted in the origins. (B) Right lateral view of SR and MR. (C) Ventral view of m. inferior rectus (IR), m. lateral rectus (LR) and IO. (D) Posterior view, with mm. oblique deflected, eyestalk (Eyt) and SR removed to illustrate insertion. Scale bar: 10 mm. II, optic nerve (cranial nerve II); IV, trochlear nerve (cranial nerve IV).

Figure 2.

Figure 2

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Left eyeball and extraocular muscles arrangement from Gymnura altavela. (A) Dorsal view of m. superior rectus (SR), m. medial rectus (MR) and m. superior oblique (SO). (B) Same view with m. inferior oblique (IO) and SO deflected, demonstrating MR insertion and innervation of SO. (C) Ventral view of IO, m. lateral rectus (LR) and m. inferior rectus (IR). (D) Same view with IO deflected, demonstrating IR insertion and innervation of SO. Scale bar: 10 mm; *eyestalk attachment point. Eyt, eyestalk; II, optic nerve (cranial nerve II); IV, trochlear nerve (cranial nerve IV).

Figure 3.

Figure 3

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Left eyeball and extraocular muscles arrangement from Pteroplatytrygon violacea. (A) Dorsal view of m. superior oblique (SO) and m. superior rectus (SR). (B) Same view with m. inferior oblique (IO) deflected, demonstrating the m. medial rectus (MR) insertion, its relation with the optic nerve (II) and eyestalk (Eyt). (C) Same in lateral view, IO and their relation with eyeball. (D) Same, IO deflected, demonstrating their relation with the optic nerve and Eyt in posterior view. Scale bar: 20 mm. IR, m. inferior rectus; LR, m. lateral rectus.

Figure 4.

Figure 4

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Right eyeball and extraocular muscles arrangement from Mobula thurstoni. (A) Anterior view of all muscles, m. lateral rectus β (LRβ) removed and arrow indicating the continuity of the oblique muscle insertion. (B) Same in posterior view, muscles deflected, demonstrating its relation with the optic nerve (II) and eyestalk (Eyt). (C) Dorsal view in situ, chondrocranium (CR) partially removed, showing its relation with LRβ. (D) Same, LRβ removed to show the LR with insertion partially removed from the ocular bulb (OB) and deflected upwards. (E) Posterior view in situ, showing the innervation of m. inferior oblique (IO) by the oculomotor nerve (III) and m. superior oblique (SO) by the trochlear nerve (IV). Scale bar: 30 mm. IR, m. inferior rectus; MR, m. medial rectus; SR, m. superior rectus.

Figure 6.

Figure 6

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Spatial relations between extraocular muscles of left eye, orbit and the line of action of the m. superior rectus (SR) and m. superior oblique (SO), and its arrangement with (CR) Chondrocranium, SR, m. lateral rectus (LR), m. lateral rectus β (LRβ), m. medial rectus (MR) and SO in dorsal view. (A) Pteroplatytrygon violacea, (B) Dasyatis hypostigma, (C) Gymnura altavela, (D) Mobula thurstoni. Species illustrations not to scale.

For each muscle, the following were analyzed: shape, origin, insertion point and fixation line. Muscle length measurements (muscle + tendon) and EYL (Ebert, 2015) were measured (calipers) and recorded (mm). To compare muscle length among species, EYL was normalized to 100 for all species. Lengths are represented as % of EYL as described (Oliva, 1967). Data were analyzed by one‐way anova, and means compared by the Tukey test (5% probability). In addition, a principal component analysis (PCA) was used to determine relationships between muscle length and species (Manly, 1986). All statistical analyses were done with r software (R Core Team, 2015).

Results

Extrinsic muscles were located completely within the orbit in all species. In D. hypostigma (Fig. 1A–D), G. altavela (Fig. 2A–D) and P. violacea (Fig. 3A–D), there were four recti (SR, LR, IR, MR) and two oblique muscles (SO and IO). However, mm. rectus in M. thurstoni (Figs 4A–E and 5) differed from the other three species in having a fifth muscle associated with LR, named here as LRβ (Figs 4C and 5). Furthermore, oblique muscles in M. thurstoni (Figs 4A–B and 5: SO and IO) were completely fused.

Figure 5.

Figure 5

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Right extraocular muscles from Mobula thurstoni, with ocular bulb and muscles partially removed, keeping their origins to demonstrate its arrangement. Scale bar: 40 mm. Eyt, eyestalk; II, optic nerve (cranial nerve II); IO, m. inferior oblique; IR, m. inferior rectus; LR, m. lateral rectus; LRβ, m. lateral rectus β; MR, m. medial rectus; SO, m. superior oblique; SR, m. superior rectus.

All recti originated posterior to the orbit and were oriented in a dorsocaudal direction. All oblique originated at that anterior region of the orbit. The IO followed the orbit dorsally, whereas the SO followed the mediorostral region. Gymnura altavela and D. hypostigma had comparable eyeballs, with the former being slightly smaller (Table 1). In P. violacea, the eyeball was smaller than M. thurstoni but bigger than the other two species (Table 1). Furthermore, there were differences between M. thurstoni and the other three species regarding angulation of SO and SR in relation to the midline (Fig. 6). The SR angle ranged from slightly smaller or larger than the SO angle with an almost equilateral shape or much larger, giving a scalene shape (Fig. 6). Axes 1 and 2 from the PCA explained 98.47% of data variability (Fig. 7). The first axis was associated with the rectus muscles and the second with oblique muscles (Table 2). The G. altavela juveniles had the lowest values, whereas they were highest for M. thurstoni, and were intermediate for three adult G. altavela specimens, D. hypostigma and P. violacea (Fig. 7). Muscle length differed significantly among sampled species (Fig. 8).

Table 1.

EYL, extraocular muscles length and width in studied rays (10 individuals for each species)

Gymnura altavela Dasyatis hypostigma Pteroplatytrygon violacea Mobula thurstoni
EYL (mm) 12.8 ± 2.3 13.9 ± 0.8 28.5 ± 1.3 31.1 ± 1.4
Length (mm)
SR 15.3 ± 7.7 17.3 ± 0.6 17.2 ± 1.4 46.7 ± 0.8
LR 19.8 ± 5.4 20.2 ± 0.7 26.5 ± 0.5 57.2 ± 0.6
IR 17.5 ± 1.5 24.1 ± 0.8 24.5 ± 0.7 55.2 ± 1.1
MR 19.0 ± 6.0 23.4 ± 0.7 21.6 ± 0.5 63.2 ± 1.1
IO 13.0 ± 3.9 24.4 ± 0.7 26.4 ± 0.7 32.8 ± 0.9
SO 11.8 ± 5.8 25.5 ± 0.8 22.7 ± 1.1 32.4 ± 0.9
LRβ − − − 27.1 ± 1.1
Width (mm)
SR 4.4 ± 0.9 6.3 ± 0.5 7.3 ± 0.8 12.1 ± 0.6
LR 3.7 ± 1.1 6.4 ± 0.5 6.0 ± 0.0 14.1 ± 0.7
IR 3.7 ± 0.6 5.7 ± 0.5 5.0 ± 1.0 10.8 ± 0.6
MR 4.2 ± 1.3 4.5 ± 0.5 5.0 ± 0.0 8.2 ± 0.6
IO 5.2 ± 2.4 15.3 ± 0.6 10.9 ± 0.6 15.1 ± 0.9
SO 9.5 ± 2.6 15.0 ± 0.6 16.6 ± 0.7 17.2 ± 0.6
LRβ − − − 8.4 ± 0.8
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EYL, eye length; IO, m. inferior oblique; IR, m. inferior rectus; LR, m. lateral rectus; LRβ, m. lateral rectus β; MR, m. medial rectus; SO, m. superior oblique; SR, m. superior rectus. Mean ± SD in mm.

Figure 7.

Figure 7

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The first and second principal component scores of extraocular muscles for Dasyatis hypostigma (purple), Gymnura altavela (red), Mobula thurstoni (green) and Pteroplatytrygon violacea (gray) generated by principal component analysis (PCA). Species illustrations not to scale.

Table 2.

Eigenvectors, eigenvalues and percentage of variance of the PCA from extraocular muscles for studied rays

Axis 1 Axis 2
SR 0.42 0.23
LR 0.41 0.32
IR 0.42 0.21
MR 0.42 0.30
IO 0.38 −0.58
SO 0.38 −0.60
Autov. 5.35 0.55
% Var. 89.22 9.25
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IO, m. inferior oblique; IR, m. inferior rectus; LR, m. lateral rectus; MR, m. medial rectus; SO, m. superior oblique; SR, m. superior rectus.

Figure 8.

Figure 8

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Mean and IC 95% of extraocular muscles length represented as a percentage (%) of eye length (EYL) for Dasyatis hypostigma (Da), Gymnura altavela (Gy), Mobula thurstoni (Mo) and Pteroplatytrygon violacea (Pt). (A) m. Superior rectus (SR); (B) m. inferior rectus (IR); (C) m. medial rectus (MR); (D) m. lateral rectus (LR); (E) m. inferior oblique (IO); (F) m. superior oblique (SO). Different letters (z, w, k and y) indicate significant differences (Tukey test, following anova).

Extraocular muscles

SR

The SR was triangular in D. hypostigma (Figs 1A and B, and 6B), but fusiform in other species. The insertion point was posterior to the EB in G. altavela (Figs 2A and 6C) and D. hypostigma, but anterior to EB in P. violacea (Figs 3C and D, and 6A) and M. thurstoni (Figs 4A–D and 6). The ITD fixation line varied among all four species: straight in G. altavela; concave in D. hypostigma; convex in P. violacea; and oblique in M. thurstoni. There were no differences in muscle length between D. hypostigma and G. altavela. The highest proportions were in M. thurstoni, whereas P. violacea had the lowest (Fig. 8A; one‐way anova, F 3,36 = 41.9; P < 0.01).

IR

The IR was fusiform in all species. The insertion point was ventral to the EB and adjacent to the IO insertion in G. altavela (Fig. 2C and D) and M. thurstoni (Figs 4A, B and D, and 5), whereas in D. hypostigma (Fig. 1C) and P. violacea (Fig. 3D) it was between the optical nerve and IO insertion. The ITD was set on an oblique fixation line in G. altavela and P. violacea, whereas D. hypostigma and M. thurstoni had a concave one. The highest proportions without significant differences were between D. hypostigma and M. thurstoni, whereas P. violacea and G. altavela had the lowest and intermediate proportions, respectively (Fig. 8B; one‐way anova, F 3,36 = 184.1; P < 0.01).

MR

The shape of MR was triangular in G. altavela (Figs 2A and B, and 6C) but fusiform in other species. The insertion point was ventral to the EB and adjacent to the oblique muscle insertion in M. thurstoni (Figs 4A–C and E, 5 and 6D), whereas in other species it was between the optical nerve and SO insertion. In G. altavela the ITD had a concave fixation line, but in the other species it set an oblique fixation line. Muscle length varied significantly between all species. Proportions were lower in P. violacea (Figs 3A and C, and 6A) followed by G. altavela, D. hypostigma (Figs 1A and D, and 6B) and larger in M. thurstoni (Fig. 8C; one‐way anova, F 3,36 = 216.6; P < 0.01).

LR

The shape of LR was triangular in M. thurstoni (Figs 4A–D, 5 and 6D) and fusiform for the other three species. The insertion point was posterior to the EB in D. hypostigma (Figs 1B–D and 6B) and P. violacea (Figs 3D and 6A); at EB in G. altavela (Figs 2C–D and 6C); and anterior to EB in M. thurstoni. The ITD set an oblique fixation line in G. altavela and D. hypostigma, concave in P. violacea and convex in M. thurstoni. There was no difference in muscle length between D. hypostigma and G. altavela. The highest proportion was in M. thurstoni and the lowest was in P. violacea (Fig. 8D; one‐way anova, F 3,36 = 153.3; P < 0.01).

LRβ in M. thurstoni

The LRβ in M. thurstoni was fusiform in shape. The insertion point was anterior to the EB and on the top of the LR insertion. The ITD set an oblique fixation line (Figs 4C, 5 and 6D).

SO and IO

The SO and IO had a strap shape, and the ITD set a convex fixation line in D. hypostigma (Figs 1A–D and 6B) and P. violacea (Figs 3A–D and 6A), whereas in G. altavela (Figs 2A–D and 6C) it was an oblique one. The IO insertion point was posterior to the EB. The SO insertion point was posterior to the EB in G. altavela and D. hypostigma, and anterior to the EB in P. violacea. The SO and IO in M. thurstoni (Figs 4A–C and 5) were completely fused in that origin (Fig. 5) and insertion (see arrow in Fig. 4A), but innervated separately by nerve IV and nerve III, respectively (Fig. 4E). The insertion point lay upon the EB at the orbit′s anterior border. The ITD set a convex fixation line. The highest values for SO and IO were in D. hypostigma (Fig. 8E and F). There was no significant difference in SO and IO between G. altavela and the other two species (Fig. 8E and F). In M. thurstoni, both SO and IO were higher than in P. violacea (Fig. 8E and F; one‐way anova: SO, F 3,36 = 88.6; P < 0.01; IO, F 3,36 = 146; P < 0.01).

Discussion

Positions and innervation patterns of extraocular muscles among the four species were in agreement with the literature (Daniel, 1934; Harder, 1975; Walker & Homberger, 1992). There are only a few studies describing the anatomy of extraocular muscles in elasmobranchs, including Von Bonde (1933), Edgeworth (1935), Oliva (1967), Isomura (1981), Miyake (1988), Nishida (1990), Gomes et al. (1991), Shirai (1992), Walker & Homberger (1992) and Lima et al. (1997). The described pattern, with six muscles (four rectus and two oblique) named according to their insertion points, was observed for D. hypostigma, G. altavela and P. violacea (Harder, 1975; Romer & Parsons, 1985; Goodrich, 1986; Nishida, 1990; Walker & Homberger, 1992; Liem & Summers, 1999). However, M. thurstoni differed from the other species by having: (i) LRβ (Figs 4C and 5); and (ii) complete fusion of oblique muscles (Figs 4A and B, and 5). The nomenclature for LRβ followed the pattern suggested by Shirai (1992), who described and named it the m. obliquus inferior β, a seventh muscle near the m. obliquus inferior present in pristiophorids, pristoids, rhinobatoids and some rajoids.

Nishida (1990), in his extensive work on Myliobatoidei phylogeny, studied the extraocular muscles of five ingroup species: Hexatrygon longirostra, Urolophus maculatus, D. brevis, Myliobatis aquila and Manta birostris; and four outgroup species: Rhinoraja longicauda, Trygonorhina fasciata, Narcine brasiliensis and Pristis microdon. The author did not describe the presence of a seventh muscle or a completely fused oblique muscle in any specimen of the group. However, D. brevis was reported to have oblique muscles ‘divided into two parts’ (sic.), which are the fused origin and the separated insertions (Nishida, 1990: fig. 44C). A similar condition was reported in D. pastinaca and Torpedo torpedo (Oliva, 1967).

Nishida (1990), in his extensive work on Myliobatoidei phylogeny, studied the extraocular muscles of five ingroup species: Hexatrygon longirostra, Urolophus maculatus, D. brevis, Myliobatis aquila and Manta birostris; and four outgroup species: Rhinoraja longicauda, Trygonorhina fasciata, Narcine brasiliensis and Pristis microdon. The author did not describe the presence of a seventh muscle or a completely fused oblique muscle in any specimen of the group. However, D. brevis was reported to have oblique muscles ‘divided into two parts’ (sic.), which are the fused origin and the separated insertions (Nishida, 1990: fig. 44C). A similar condition was reported in D. pastinaca and Torpedo torpedo (Oliva, 1967).

Fossil, morphological and molecular data supported the hypothesis that mobulids are one of the most derived groups of elasmobranchs and closely related to rhinopterids (cownose rays, genus Rhinoptera) within a polyphyletic clade of Myliobatidae (Nishida, 1990; Lovejoy, 1996; McEachran et al. 1996; Shirai, 1996; Dunn et al. 2003; De Carvalho et al. 2004; McEachran & Aschliman, 2004; Claeson et al. 2010; Aschliman et al. 2012; Naylor et al. 2012). Thus, the completely fused oblique muscles in M. thurstoni represented a condition derived from that with just the fused origin and separated insertions for D. brevis, D. pastinaca and T. torpedo (Oliva, 1967; Nishida, 1990: fig. 44C), but apparently autapomorphic for that single examined species of Mobula. Such characteristics should be considered in future phylogenetic studies, ideally with inclusion of additional species.

Evidence of up to seven extraocular muscles and the condition of two abducens‐innervated eye muscles in placoderms (as an outgroup of all extant gnathostomes) would support homology between those of the lamprey and the external rectus and retractor bulbi of tetrapods (Fritzsch et al. 1990; Young, 2008).

The accessory lateral rectus muscle (aLR) is a well‐known abducens‐innervated muscle, with a potential role in strabismus in monkeys (Spencer & Porter, 1981; Schnyder, 1984; Boothe et al. 1990; Narasimhan et al. 2007) and humans (Von Lüdinghausen et al. 1999; Liao & Hwang, 2014). Typically, the aLR is very small, inconsistently located, often superior to the optic nerve, medial to the LR and inserts at the posterior half of the eye ball, approximately between LR and SR (Schnyder, 1984: fig. 1; Liao & Hwang, 2014).

Schnyder (1984) proposed that the aLR represented a residual retractor bulbi (RB) from lower vertebrates, but Narasimhan et al. (2007) refuted it by comparing their origins, because aLR originates on the LR and in lower vertebrates, the RB has four broad heads originating at the optic foramen.

According to Narasimhan et al. (2007), the aLR originated on the orbital surface of the deep LR belly in monkeys. Liao & Hwang (2014) described a similar condition in humans, with aLR and LR originating from the same tendon, and that it may have a muscle belly or exist simply as a fibrous band. Both descriptions implied that aLR does not have its own source, but that it actually appeared as an appendix LR (accessory). Both aLR and LR were equally innervated although, in aLR, innervation was to single fibers (Schnyder, 1984).

The LRβ in M. thurstoni was similar to topography of mammalian aLR. Notwithstanding the similarites, these muscles are considered non‐homologous, as LRB was larger and had its own origin. Although innervation of LRβ was not confirmed, due to the proximity of its origin with the LR, it was probably innervated by the same nerve (VI). Future studies are needed to confirm its innervation in M. thurstoni and to determine whether LRβ is present in any other Myliobatiformes.

The origin of recti and oblique muscles in the four species studied was posterior and anterior to the orbit, respectively, in agreement with Nishida (1990). The geometry of extraocular muscles in frontal‐ and lateral‐eyed animals (man, cat, guinea pig and rabbit) was examined by orientation of semicircular canals; there were marked differences in insertion points and line action angulation of the superior muscles (SR and SO) on the globe between them (Simpson & Graf, 1981). Although semicircular canals have apparently not been studied in rays, the pattern of insertion and muscle path had considerable modifications, with apparent associations to muscle function and animal habitat. Modified insertions and muscle path of vertical extraocular muscles were indicative of requirements for producing specific compensatory eye movements, and resulted in disparate secondary kinematic actions. The muscle paths of SO and SR in M. thurstoni resembled the rabbit, with the ray eyes positioned more laterally, similar to that species. However, in the three other species, muscle paths resembled the guinea pig, with eyes positioned slightly forward compared with other species (Simpson & Graf, 1981: 23; fig. 1). Comparing the angle among studied ray species, rabbit and guinea pig, the SR tended to form an acute angle (56–65 °). However, in humans and cats, this angle tends to be obtuse (23–25 °), almost parallel to the body axis, in species with eyes strongly positioned forward.

The association of recti muscles on the first axis and oblique muscles on the second axis of the PCA (Fig. 8) indicated the importance of the size of the recti muscles to these rays, as the bottom‐dwelling G. altavela juveniles had smaller negative values, whereas pelagic M. thurstoni had higher positive values. Three G. altavela individuals, D. hypostigma both bottom‐dwelling rays and pelagic P. violacea remained in the middle of the PCA axis with negative values, separated without overlap. Furthermore, the slightly higher values and close position to D. hypostigma in Axis 1, P. violacea had shorter extraocular muscle lengths related to eyeball diameter compared with the other species. Variation in muscle length in G. altavela was attributed to a combination of juveniles and adults. These differences in muscle path angulation and size of recti and oblique muscles have a role in eyeball rotation, providing different visual fields among studied species (McComb & Kajiura, 2008).

In Myliobatiformes, the pectoral girdle has four distinct articular regions constituted by three condyles and a facet (Da Silva & De Carvalho, 2015) and a diverse general locomotor behavior (Rosenberger, 2001). Stingrays such as Dasyatis species have pectoral fin skeletal structures with reduced calcification and joint staggering (Schaefer & Summers, 2005); they move with an undulatory swimming mode (Rosenberger, 2001) and feed mainly on benthic prey (Jacobsen & Bennett, 2013). The joint‐staggering pattern was lost in P. violacea, and the chains of calcification on its robust radials are very broad and highly mineralized (Schaefer & Summers, 2005) allowing an oscillatory swimming mode (Rosenberger, 2001). These changes allow it to move away from the bottom to open, clear water. The oscillatory swimming mode and abiotic factors such as light and visibility could have allowed new angles to the visual field and favored the development of its eyeball, leading to a shift in the kind of prey captured. It feeds on fish, cephalopods, pteropods and small crustaceans (Véras et al. 2009; Jacobsen & Bennett, 2013). Despite extraocular muscles in P. violacea and D. hypostigma being smaller and having similar sizes (Table 1) when related to EYL (Fig. 3), comparatively, the P. violacea values are smaller, as its EYL is almost twice that of D. hypostigma.

The butterfly rays (Gymnuridae) have a crustal calcification pattern on pectoral fin skeletal structure with cross‐bracing radials (Schaefer & Summers, 2005). Its semi‐oscillatory swimming mode allows both undulate/bottom and oscillatory/column water swimming (Rosenberger, 2001) or delivery of physical blows to stun prey before ingestion in an ambush feeding strategy (Jacobsen et al. 2009; Jacobsen & Bennett, 2013). Gracile and flexible jaws with large gape allow engulfment of food much larger than the resting mouth opening (Dean et al. 2007). Furthermore, M. thurstoni shares the same crustal calcification pattern with cross‐bracing radials (Schaefer & Summers, 2005), but with an oscillatory mobuliform swimming mode that consists of vertical flapping movements of the pectoral fins, similar to the flight of birds (Rosenberger & Westneat, 1999; Wilga & Lauder, 2004), and a filter‐feeding behavior preying primarily on Euphausiids and Mysids (Notarbartolo‐di‐Sciara, 1988; Schaefer & Summers, 2005; Adnet et al. 2012). In this study, bottom‐dwelling rays had smaller eyes whereas pelagic had the larger ones, in agreement with the literature (Lisney & Collin, 2007).

Morphologically, M. thurstoni had larger, laterally placed eyes (laterally placed as well as Rhinoptera bonasus). Based on a fossil‐calibrated Bayesian random local clock analysis, it is estimated that mobulids diverged from Rhinoptera ~ 30 Mya (Poortvliet et al. 2015). According to McComb & Kajiura (2008), R. bonasus demonstrated a 360 ° panoramic view in vertical plane with large anterior binocular 46 ° overlaps, resutling in large posterior blind areas. It is expected that M. thurstoni has a monocular visual field frontally occluded by cephalic fins but with a panoramic view in a vertical plane. This view is exercised by synergistic movements of muscles attached to the anterior (SO and IO) and posterior (SR, LR and LRβ) regions of the eyeball. The increasing visual perception capacity of the environment facilitates rapid detection of predators, creation of escape routes and improves foraging for food, as upwelling areas concentrate resources in a relatively small geographic area, often providing densities of food particles high enough to meet the large energy demands of filter‐feeding marine megafauna (Croll et al. 2005).

Extraocular muscles differed in shape, insertion point and form of the line described by the ITDs and length among the four studied species. Although it was not possible to establish a pattern, these anatomical differences should facilitate grouping the studied species according to their life history. The presence of LRβ and fusion of oblique muscle in other Mobulid species need to be investigated. Extraocular muscles were not extraordinarily uniform in all vertebrates and, therefore, could be used in future comparative anatomical, functional and evolutionary studies in various species that, until now, have not been well characterized.

Acknowledgements

The authors thank: Ulisses L. Gomes (UERJ), Marcelo R. de Carvalho (IB USP), Arani N. B. Mariana and Pedro P. Bombonato (UFMV USP) for inspiration and extensive discussions; Marcelo Machado (UFPR) and André Casas (UFAC) for help with dissection and extensive discussions regarding results; and two anonymous reviewers who suggested substantial revisions. The authors also thank Dr John Kastelic, University of Calgary, Alberta, Canada, for English edition on manuscript. This work was partially supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil) to C.M.C. by a doctorate grant and post‐doc grant (proc. #8739/13‐7).

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