Journal of Anatomy

J. Anat. (2016) 228, pp877--886 doi: 10.1111/joa.12438

REVIEW ARTICLE

Comparative anatomy of the extraocular muscles in

four Myliobatoidei rays (Batoidea, Myliobatiformes)

R. Kfoury Jr4
Carlo M. Cunha,1,2 Luciano E. Oliveira3 and Jose
1
Academy of Natural Sciences, Philadelphia, PA, USA
2
Capes Foundation, Ministry of Education of Brazil, Bras
ılia, DF, Brazil
3
Ecology and Environmental Resources Post Graduation Program, Uberla ^ ndia Federal University, Uberla
^ndia, MG, Brazil
4
Department of Surgery, School of Veterinary Medicine and Animal Science, University of Sa ~o Paulo, SP, Sa
~o Paulo, Brazil

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 b (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.
Key words: Dasyatis; Gymnura; Mobula; morphology; Pteroplatytrygon.

Elasmobranchs have a variety of advanced visual features,

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 gener-
ally described as having small eyes and poor vision, relying
primarily on other senses (e.g. smell, hearing and electrore-
ception). Nevertheless, after discovery of their double retina
containing cones and rods (Gilbert, 1963; Gruber et al.
1963; Hamasaki & Gruber, 1965; Cohen, 1989), more evi-
dence 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).
Correspondence
Carlo Magenta Cunha, Academy of Natural Sciences, 1900 Benjamin
Franklin Parkway, Philadelphia, PA 19103, USA.
E: carlomagenta@gmail.com
Accepted for publication 16 December 2015
Article published online 8 February 2016
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 previ-
ously 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 ben-
thopelagic 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 (Hair-
ston et al. 1982). In fish, eye morphology can vary, depend-
ing 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 move-
ment, and are regarded as constant in number, arrange-
ment and innervation in vertebrates (Goodrich, 1986).

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878 Anatomy extraocular muscles Myliobatoidei rays, C. M. Cunha et al.

There are four rectus and two oblique muscles (Nomina

A
Anatomica Veterinaria, 2007); during eyeball movement,
these muscles form three pairs (antagonistic and synergis- B
tic). 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 responsi- D
ble for rotation of the eyeball around the optical axis
(Harder, 1975; Carpenter, 1977). The innervation pattern C
by three cranial nerves is consistent among extant verte-
brates (Fritzsch et al. 1990). Nerve IV innervates superior
oblique. Nerve VI innervates lateral rectus in chon-
drichthyan 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 Fig. 1 Right eyeball (flipped horizontally) and extraocular muscles
study was to characterize extraocular muscles in four Mylio- arrangement from Dasyatis hypostigma. (A) Dorsal view of m. superior
batoidei ray species from diverse habitats with divergent rectus (SR), m. medial rectus (MR) and mm. oblique (SO and IO) with
behaviors. The hypothesis that there are significant differ- part of the chondrocranium (CR) attachted in the origins. (B) Right lat-
eral view of SR and MR. (C) Ventral view of m. inferior rectus (IR), m.
ences among species in extraocular muscles morphometry
lateral rectus (LR) and IO. (D) Posterior view, with mm. oblique
was tested. It was expected that results could provide a new
deflected, eyestalk (Eyt) and SR removed to illustrate insertion. Scale
basis for future evolutionary/phylogenetic studies and com- bar: 10 mm. II, optic nerve (cranial nerve II); IV, trochlear nerve (cranial
parative homology within myliobatoids. nerve IV).

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 vio-
lacea; 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 trawl-
~o Paulo state,
ing and longline fishing vessels off the coast of Sa
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 Veterina
ria da Universidade de Sa ~o 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; LRb, m. lateral rectus b; 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 mus-
cles in Figs 2–4 and 6.
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 deter-
mine 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) dif-
fered from the other three species in having a fifth muscle
associated with LR, named here as LRb (Figs 4C and 5). Fur-
thermore, oblique muscles in M. thurstoni (Figs 4A–B and
5: SO and IO) were completely fused.
All recti originated posterior to the orbit and were ori-
ented in a dorsocaudal direction. All oblique originated at

© 2016 Anatomical Society


A B
C D
Fig. 2 Left eyeball and extraocular muscles arrangement from Gym-
nura altavela. (A) Dorsal view of m. superior rectus (SR), m. medial
rectus (MR) and m. superior oblique (SO). (B) Same view with m. infe-
rior 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 attach-
ment point. Eyt, eyestalk; II, optic nerve (cranial nerve II); IV, trochlear
nerve (cranial nerve IV).
A B
C D
Fig. 3 Left eyeball and extraocular muscles arrangement from Ptero-
platytrygon 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 rela-
tion 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.
© 2016 Anatomical Society
Anatomy extraocular muscles Myliobatoidei rays, C. M. Cunha et al. 879
A B
C D
Fig. 4 Right eyeball and extraocular muscles arrangement from Mob-
ula thurstoni. (A) Anterior view of all muscles, m. lateral rectus b (LRb)
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 LRb. (D) Same, LRb removed to show the LR with insertion par-
tially 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.
Fig. 5 Right extraocular muscles from Mobula thurstoni, with ocular
bulb and muscles partially removed, keeping their origins to demon-
strate 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; LRb, m. lateral rectus b; MR, m. medial rectus; SO, m.
superior oblique; SR, m. superior rectus.
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
880 Anatomy extraocular muscles Myliobatoidei rays, C. M. Cunha et al.

Table 1 EYL, extraocular muscles length and width in studied rays Axes 1 and 2 from the PCA explained 98.47% of data vari-
(10 individuals for each species). ability (Fig. 7). The first axis was associated with the rectus
muscles and the second with oblique muscles (Table 2). The
Gymnura Dasyatis Pteroplatytrygon Mobula
G. altavela juveniles had the lowest values, whereas they
altavela hypostigma violacea thurstoni
were highest for M. thurstoni, and were intermediate for
three adult G. altavela specimens, D. hypostigma and
EYL (mm) 12.8
2.3 13.9
0.8 28.5
1.3 31.1
1.4
Length (mm) P. violacea (Fig. 7). Muscle length differed significantly
SR 15.3
7.7 17.3
0.6 17.2
1.4 46.7
0.8 among sampled species (Fig. 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 Extraocular muscles
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 SR
LRb    27.1
1.1
Width (mm) The SR was triangular in D. hypostigma (Figs 1A and B, and
SR 4.4
0.9 6.3
0.5 7.3
0.8 12.1
0.6 6B), but fusiform in other species. The insertion point was
LR 3.7
1.1 6.4
0.5 6.0
0.0 14.1
0.7 posterior to the EB in G. altavela (Figs 2A and 6C) and
IR 3.7
0.6 5.7
0.5 5.0
1.0 10.8
0.6 D. hypostigma, but anterior to EB in P. violacea (Figs 3C
MR 4.2
1.3 4.5
0.5 5.0
0.0 8.2
0.6 and D, and 6A) and M. thurstoni (Figs 4A–D and 6). The ITD
IO 5.2
2.4 15.3
0.6 10.9
0.6 15.1
0.9
fixation line varied among all four species: straight in
SO 9.5
2.6 15.0
0.6 16.6
0.7 17.2
0.6
G. altavela; concave in D. hypostigma; convex in P. violacea;
LRb    8.4
0.8
and oblique in M. thurstoni. There were no differences in
EYL, eye length; IO, m. inferior oblique; IR, m. inferior rectus; LR, muscle length between D. hypostigma and G. altavela. The
m. lateral rectus; LRb, m. lateral rectus b; MR, m. medial rectus; highest proportions were in M. thurstoni, whereas
SO, m. superior oblique; SR, m. superior rectus. Mean
SD in mm. P. violacea had the lowest (Fig. 8A; one-way ANOVA,
F3,36 = 41.9; P < 0.01).
eyeballs, with the former being slightly smaller (Table 1). In
P. violacea, the eyeball was smaller than M. thurstoni but
IR
bigger than the other two species (Table 1). Furthermore,
there were differences between M. thurstoni and the other The IR was fusiform in all species. The insertion
three species regarding angulation of SO and SR in relation point was ventral to the EB and adjacent to the IO insertion
to the midline (Fig. 6). The SR angle ranged from slightly in G. altavela (Fig. 2C and D) and M. thurstoni (Figs 4A,
smaller or larger than the SO angle with an almost equilat- B and D, and 5), whereas in D. hypostigma (Fig. 1C) and
eral shape or much larger, giving a scalene shape (Fig. 6). P. violacea (Fig. 3D) it was between the optical nerve and

Fig. 6 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 b (LRb), 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.

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 Anatomy extraocular muscles Myliobatoidei rays, C. M. Cunha et al. 881

Fig. 7 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 LR

PCA from extraocular muscles for studied rays.
The shape of LR was triangular in M. thurstoni (Figs 4A–D,
Axis 1 Axis 2 5 and 6D) and fusiform for the other three species. The
insertion point was posterior to the EB in D. hypostigma
SR 0.42 0.23 (Figs 1B–D and 6B) and P. violacea (Figs 3D and 6A); at EB
LR 0.41 0.32 in G. altavela (Figs 2C–D and 6C); and anterior to EB in
IR 0.42 0.21 M. thurstoni. The ITD set an oblique fixation line in G. al-
MR 0.42 0.30
tavela and D. hypostigma, concave in P. violacea and con-
IO 0.38 0.58
SO 0.38 0.60
vex in M. thurstoni. There was no difference in muscle
Autov. 5.35 0.55 length between D. hypostigma and G. altavela. The highest
% Var. 89.22 9.25 proportion was in M. thurstoni and the lowest was in P. vi-
olacea (Fig. 8D; one-way ANOVA, F3,36 = 153.3; P < 0.01).
IO, m. inferior oblique; IR, m. inferior rectus; LR, m. lateral
rectus; MR, m. medial rectus; SO, m. superior oblique; SR,
m. superior rectus. LRb in M. thurstoni
The LRb in M. thurstoni was fusiform in shape. The insertion
point was anterior to the EB and on the top of the LR inser-
IO insertion. The ITD was set on an oblique fixation line tion. The ITD set an oblique fixation line (Figs 4C, 5 and 6D).
in G. altavela and P. violacea, whereas D. hypostigma
and M. thurstoni had a concave one. The highest
SO and IO
proportions without significant differences were between
D. hypostigma and M. thurstoni, whereas P. violacea and The SO and IO had a strap shape, and the ITD set a con-
G. altavela had the lowest and intermediate proportions, vex fixation line in D. hypostigma (Figs 1A–D and 6B) and
respectively (Fig. 8B; one-way ANOVA, F3,36 = 184.1; P < 0.01). 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
MR
posterior to the EB in G. altavela and D. hypostigma, and
The shape of MR was triangular in G. altavela (Figs 2A and anterior to the EB in P. violacea. The SO and IO in
B, and 6C) but fusiform in other species. The insertion point M. thurstoni (Figs 4A–C and 5) were completely fused in
was ventral to the EB and adjacent to the oblique muscle that origin (Fig. 5) and insertion (see arrow in Fig. 4A), but
insertion in M. thurstoni (Figs 4A–C and E, 5 and 6D), innervated separately by nerve IV and nerve III, respectively
whereas in other species it was between the optical nerve (Fig. 4E). The insertion point lay upon the EB at the orbit0 s
and SO insertion. In G. altavela the ITD had a concave fixa- anterior border. The ITD set a convex fixation line. The
tion line, but in the other species it set an oblique fixation highest values for SO and IO were in D. hypostigma (Fig. 8E
line. Muscle length varied significantly between all species. and F). There was no significant difference in SO and IO
Proportions were lower in P. violacea (Figs 3A and C, and between G. altavela and the other two species (Fig. 8E and
6A) followed by G. altavela, D. hypostigma (Figs 1A and D, F). In M. thurstoni, both SO and IO were higher than in
and 6B) and larger in M. thurstoni (Fig. 8C; one-way ANOVA, P. violacea (Fig. 8E and F; one-way ANOVA: SO, F3,36 = 88.6;
F3,36 = 216.6; P < 0.01). P < 0.01; IO, F3,36 = 146; P < 0.01).

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882 Anatomy extraocular muscles Myliobatoidei rays, C. M. Cunha et al.
Discussion
Positions and innervation patterns of extraocular muscles
among the four species were in agreement with the litera-
ture (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 spe-
cies by having: (i) LRb (Figs 4C and 5); and (ii) complete
fusion of oblique muscles (Figs 4A and B, and 5). The
nomenclature for LRb followed the pattern suggested by
Shirai (1992), who described and named it the m. obliquus
Fig. 8 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).
inferior b, 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 fasci-
ata, Narcine brasiliensis and Pristis microdon. The author
did not describe the presence of a seventh muscle or a com-
pletely 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 sim-
ilar 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
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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 repre-
sented a condition derived from that with just the fused ori-
gin and separated insertions for D. brevis, D. pastinaca and
T. torpedo (Oliva, 1967; Nishida, 1990: fig. 44C), but appar-
ently 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 con-
dition of two abducens-innervated eye muscles in placo-
derms (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 stra-
bismus in monkeys (Spencer & Porter, 1981; Schnyder, 1984;
Boothe et al. 1990; Narasimhan et al. 2007) and humans
(Von Lu € dinghausen et al. 1999; Liao & Hwang, 2014). Typi-
cally, 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 ori-
gins, because aLR originates on the LR and in lower verte-
brates, 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 LRb 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 LRb 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 LRb 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 extraoc-
© 2016 Anatomical Society
Anatomy extraocular muscles Myliobatoidei rays, C. M. Cunha et al. 883
ular 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 inser-
tion points and line action angulation of the superior mus-
cles (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 mus-
cle path had considerable modifications, with apparent
associations to muscle function and animal habitat. Modi-
fied insertions and muscle path of vertical extraocular mus-
cles 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 posi-
tioned 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 obli-
que 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 val-
ues, 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. Varia-
tion in muscle length in G. altavela was attributed to a com-
bination 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 loco-
motor behavior (Rosenberger, 2001). Stingrays such as Dasy-
atis species have pectoral fin skeletal structures with
reduced calcification and joint staggering (Schaefer & Sum-
mers, 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 (Schae-
fer & 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
884 Anatomy extraocular muscles Myliobatoidei rays, C. M. Cunha et al.
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, cephalo-
pods, pteropods and small crustaceans (Ve
ras 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), compara-
tively, the P. violacea values are smaller, as its EYL is almost
twice that of D. hypostigma.
The butterfly rays (Gymnuridae) have a crustal calcifica-
tion pattern on pectoral fin skeletal structure with cross-
bracing radials (Schaefer & Summers, 2005). Its semi-oscilla-
tory 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 con-
sists 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 & Kaji-
ura (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 LRb) 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 LRb and fusion of oblique mus-
cle in other Mobulid species need to be investigated.
Extraocular muscles were not extraordinarily uniform in all
vertebrates and, therefore, could be used in future compar-
ative anatomical, functional and evolutionary studies in var-
ious 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 Andre
Casas (UFAC) for help with dissection and extensive dis-
cussions regarding results; and two anonymous reviewers who sug-
gested 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 (Coor-
~o de Aperfeicßoamento de Pessoal de N
ıvel Superior, Brazil)
denacßa
to C.M.C. by a doctorate grant and post-doc grant (proc.
#8739/13-7).
References
Adnet S, Cappetta H, Guinot G, et al. (2012) Evolutionary history
of the devilrays (Chondrichthyes: Myliobatiformes) from fossil
and morphological inference. Zool J Linn Soc 166, 132–159.
Aschliman NC, Nishida M, Miya M, et al. (2012) Body plan con-
vergence in the evolution of skates and rays (Chondrichthyes:
Batoidea). Mol Phylogenet Evol 63, 28–42.
Boothe RG, Quick M, Josse MV, et al. (1990) ALR orbital geome-
try in normal and naturally strabismic monkeys. Invest Oph-
thalmol Vis Sci 31, 1168–1174.
Carpenter RHS (1977) Movements of the Eyes. London: Pion-
London.
Casas ALS, Intelizano W, Castro MFS, et al. (2005) Nerves of the
mandibular musculature of the sand tiger shark Carcharias
taurus (Rafinesque, 1810) (Chondrichthyes: Odontaspididae).
Int J Morphol 23, 387–392.
Claeson KM, O’Leary MA, Roberts EM, et al. (2010) First Meso-
zoic record of the stingray Myliobatis wurnoensis from Mali
and a phylogenetic analysis of Myliobatidae incorporating
dental characters. Acta Palaeontol Pol 55, 655–674.
Cohen JL (1989) Functional pathways in the elasmobranch
retina. J Exp Zool 2 (Suppl), 75–82.
Compagno LJV (1990) Alternative life-history styles of cartilagi-
nous fishes in time and space. Environ Biol Fish 28, 33–75.
Croll DA, Marinovic B, Benson S, et al. (2005) From wind to
whales: trophic links in a coastal upwelling system. Mar Ecol
Prog Ser 289, 117–130.
Daniel JF (1934) The Elasmobranch Fishes, 3rd edn. Berkeley:
University of California Press.
De Carvalho MR, Maisey JG, Grande L (2004) Freshwater stin-
grays of the Green River formation of Wyoming (early
Eocene), with the description of a new genus and species and
an analysis of its phylogenetic relationships (Chondrichthyes:
Myliobatiformes). Bull Am Mus Nat Hist 284, 1–36.
Dean MN, Bizzarro JJ, Summers AP (2007) The evolution of cra-
nial design, diet, and feeding mechanisms in batoid fishes.
Integr Comp Biol 47, 70–81.
© 2016 Anatomical Society

Dunn KA, McEachran JD, Honeycutt RL (2003) Molecular phylo-
genetics of myliobatiform fishes (Chondrichthyes: Myliobati-
formes), with comments on the effects of missing data on
parsimony and likelihood. Mol Phylogenet Evol 27, 259–270.
Ebert DA (2015) Deep–sea cartilaginous fishes of the Southeast-
ern Atlantic Ocean. FAO Species Catalogue for Fishery Pur-
poses, No. 9. Rome: FAO.
Edgeworth FH (1935) The Cranial Muscles of Vertebrates. Lon-
don: Cambridge University Press.
Fanta E, Meyer AA, Gro € tzner SR, et al. (1994) Comparative study
on feeding strategy and activity patterns of two Antartic fish:
Trematomus newnesi Boulenger, 1902 and Gobionotothen
gibberifrons (Lo € nnberg, 1905) (Pisces, Nototheniidae) under
different light conditions. Antarct Rec 38, 13–29.
Figueiredo JL (1977) Manual de peixes marinhos do Sudeste do
Brasil: I Introducßa~o. Cacßo~ es, Raias e Quimeras. Sa ~o Paulo:
Museu de Zoologia da Universidade de Sa ~o Paulo.
Fritzsch B, Sonntag R, Dubuc R, et al. (1990) Organization of
the six motor nuclei innervating the ocular muscles in lam-
prey. J Comp Neurol 294, 491–506.
Gadig OBF, Namora RC, Motta FS (2003) Occurrence of the bent-
fin devil ray, Mobula thurstoni (Chondrichthyes: Mobulidae),
in the western Atlantic. J Mar Biol Assoc UK 83, 869–870.
Gilbert PW (1963) The visual apparatus of sharks. In: Sharks and
Survival. (ed. Gilbert PW), pp. 283–326. Boston: DC Heath.
Gomes UL, Santos HRS, Medina AE (1991) Anophthalmia in
Dasyatis sayi (Lesueur, 1817) (Myliobatiformes, Dasyatidae). An
Acad Bras Cienc 63, 307–313.
Goodrich SE (1986) Studies on the Structure and Development
of Vertebrates. Chicago: University of Chicago Press.
Graf W, Brunken WJ (1984) Elasmobranch oculomotor organiza-
tion: anatomical and theoretical aspects of the phylogenetic
development of vestibulo-oculomotor connectivity. J Comp
Neurol 227, 569–581.
Gruber SH, Cohen JL (1978) Visual system of the elasmobranch:
state of the art 1960–1975. In: Sensory Biology of Sharks,
Skates and Rays. (eds Hodgson ES, Mathewson RF), pp. 11–
116. Arlington: Office of Naval Research.
Gruber SH, Hamasaki DI, Bridges CDB (1963) Cones in the retina
of the lemon shark (Negaprion brevirostris). Vision Res 3, 397–
399.
Hairston N Jr, Li KT, Easter SS Jr (1982) Fish vision and the
detection of planktonic prey. Science 218, 1240–1242.
Hamasaki DI, Gruber SH (1965) The photoreceptors of the nurse
shark Ginglymostoma cirratum and the stingray, Dasyatis sayi.
Bull Mar Sci 15, 1051–1059.
Harder W (1975) Anatomy of Fishes. Sttutgart: E. Schweizer-
bart’sche verlagsbuchhandlung (Na €gele u. Obermiller).
Hart NS, Lisney TJ, Collin SP (2006) Visual communication in
elasmobranchs. In: Communication in Fishes, Vol. 1. (eds
Ladich F, Collin SP, Moller P, Kapoor BG), pp. 337–392. Enfield,
NH: Science Publishers.
Hueter RE (1991) Adaptations for spatial vision in sharks. J Exp
Zool 5, 130–141.
Hueter RE, Cohen JL (1991) Vision in elasmobranchs: a compara-
tive and ecological perspective. J Exp Zool 5(Suppl), 1–182.
Hueter RE, Mann DA, Maruska KP, et al. (2004) Sensory biology
of elasmobranchs. In: Biology of Sharks and their Relatives.
(eds Carrier JC, Musick JA, Heithaus MR), pp. 326–368. New
York: CRC Press.
Isomura G (1981) Comparative anatomy of the extrinsic do olho
muscles in vertebrates. Anat Anz 150, 498–515.
© 2016 Anatomical Society
Anatomy extraocular muscles Myliobatoidei rays, C. M. Cunha et al. 885
Jacobsen IP, Bennett MB (2013) A comparative analysis of feed-
ing and trophic level ecology in stingrays (Rajiformes; Mylio-
batoidei) and electric rays (Rajiformes: Torpedinoidei). PLoS
ONE 8, e71348.
Jacobsen IP, Johnson JW, Bennett MB (2009) Diet and reproduc-
tion in the Australian butterfly ray Gymnura australis from
northern and north-eastern Australia. J Fish Biol 75, 2475–
2489.
Liao YJ, Hwang JJ (2014) Accessory lateral rectus in a patient
with normal ocular motor control. J Neuroophthalmol 34,
153–154.
Liem KF, Summers AP (1999) Muscular system: gross anatomy and
functional morphology of muscles. In: Sharks, Skates and Rays –
the Biology of Elasmobranch Fishes. (ed. Hamlett WC), pp. 93–
114. MD: John Hopkins University Press, Baltimore, Maryland.
Lima MC, Gomes UL, Souza-Lima W, et al. (1997) Estudo ana-
^ mico comparativo da regia
to ~o cefa
lica pre
-branquial de
Sphyrna lewini (Griffith & Smith) e Rhizoprionodon lalandii
(Valenciennes) (Elasmobranchii, Carcharhiniformes) relaciona-
dos com a presencßa do cefalofo
lio em Sphyrna Refinesque.
Rev Bras Zool 14, 347–370.
Lisney TJ, Collin SP (2007) Relative eye size in elasmobranchs.
Brain Behav Evol 69, 266–279.
Lovejoy NR (1996) Systematics of myliobatoid elasmobranchs:
with emphasis on the phylogeny and historical biogeography
of neotropical freshwater stingrays (Potamotrygonidae: Raji-
formes). Zool J Linn Soc 117, 207–257.
Manly BFJ (1986) Multivariate Statistical Methods: a Primer. Lon-
don: Chapman & Hall.
McComb DM, Kajiura SM (2008) Visual fields of four batoid
fishes: a comparative study. J Exp Biol 211, 482–490.
McEachran JD, Aschliman NC (2004) Phylogeny of Batoidea. In:
Biology of Sharks and Their Relatives. (eds Carrier JC, Musick
JA, Heithaus MR), pp. 79–113. New York,NY: CRC Press.
McEachran JD, Dunn KA, Miyake T (1996) Interrelationships of
the batoid fishes (Chondrichthyes, Batoidea). In: Interrelation-
ships of Fishes. (eds Stiassny MLJ, Parenti LR, Johnson GD), pp.
63–84. San Diego,CA: Academic Press.
Menezes NA, Wagner HJ, Alf MA (1981) Retinal adaptations in
fishes from a floodplain environment in the central amazon
basin. Rev Can Biol 40, 111–132.
Miyake T (1988) The systematic of the stingray genus Urotrygon
with comments on the interrelationships within Urolophidae
(Chondrichthyes, Myliobatiformes). Texas: Texas A&M Univer-
sity Consortium Press. Unpublished Ph.D. dissertation.
Narasimhan A, Tychsen L, Poukens V, et al. (2007) Horizontal
rectus muscle anatomy in naturally and artificially strabismic
monkeys. Invest Ophthalmol Vis Sci 48, 2576–2588.
Naylor GJP, Caira JN, Jensen K, et al. (2012) Elasmobranch phy-
logeny: a mitochondrial estimate based on 595 species. In:
Biology of Sharks and their Relatives. (eds Carrier JC, Musick
JA, Heithaus MR), pp. 31–56. Boca Raton: CRC Press.
Nelson JS (2006) Fishes of the World, 4th edn. New York: John
Wiley.
Nishida K (1990) Phylogeny of the suborder Myliobatidoidei.
Mem Fac Fish Hokkaido Univ 37, 1–108.
Nomina Anatomica Veterinaria (2007) International Committee
on Veterinary Gross Anatomical Nomenclature (ICVGAN), 5th
edn. . http://www.wava-amav.org/nav_nev.htm
Notarbartolo-Di-Sciara G (1988) Natural history of the rays
of the genus Mobula in the Gulf of California. Fish Bull 86,
45–66.
886 Anatomy extraocular muscles Myliobatoidei rays, C. M. Cunha et al.
Oliva O (1967) On topography of eye muscles of several elasmo-
branchs with regard to their life habits. Acta Soc Zool Bohe-
mosl 31, 51–67.
Poortvliet M, Olsen JL, Croll DA, et al. (2015) A dated molecular
phylogeny of manta and devil rays (Mobulidae) based on
mitogenome and nuclear sequences. Mol Phylogenet Evol 83,
72–85.
Pough FH, Heiser JB, McFarland WN (1999) A Vida Dos Vertebra-
dos, 2nd edn. Sa~o Paulo: Atheneu.
R Core Team (2015) R: a Language and Environment for Statisti-
cal Computing. Vienna, Austria: R Foundation for Statistical
Computing. https://www.R-project.org
Romer AS, Parsons CM (1985) Anatomia Comparada Dos Verte-
brados. Sa~o Paulo: Atheneu.
Rosenberger LJ (2001) Pectoral fin locomotion in batoid fishes:
undulation versus oscillation. J Exp Biol 204, 379–394.
Rosenberger LJ, Westneat MW (1999) Functional morphology of
undulatory pectoral fin locomotion in the stingray, Taeniura
lymma. J Exp Biol 202, 3523–3539.
Santos HRS, Carvalho MR (2004) Description of a new species of
whiptailed stingray from the southwestern Atlantic Ocean
(Chondrichthyes, Myliobatiformes, Dasyatidae). Bol Mus Nac
NS Zool 516, 1–24.
Schaefer JT, Summers AP (2005) Batoid wing skeletal structure:
novel morphologies, mechanical implications, and phyloge-
netic patterns. J Morphol 264, 298–313.
Schnyder H (1984) The innervation of the monkey accessory lat-
eral rectus muscle. Brain Res 296, 139–144.
Shirai S (1992) Squalean Phylogeny a New Framework of “Squa-
loid” Sharks and Related Taxa. Sapporo: Hokkaido University
Press.
Shirai S (1996) Phylogenetic interrelationships of neoselachians
(Chondrichthyes: Euselachii). In: Interrelationships of Fishes.
(eds Stiassny MLJ, Parenti LR, Johnson GD), pp. 9–34. San
Diego,CA: Academic Press.
Da Silva JPCB, De Carvalho MR (2015) Morphology and phyloge-
netic significance of the pectoral articular region in elasmo-
branchs (Chondrichthyes). Zool J Linn Soc, in press. DOI:
10.1111/zoj.12287.
Simpson JI, Graf W (1981) Eye-muscle geometry and compen-
satory eye movements in lateral-eyed and frontal-eyed ani-
mals. Ann N Y Acad Sci 374, 20–30.
Spencer RF, Porter JD (1981) Innervation and structure of
extraocular muscles in the monkey in comparison to those of
the cat. J Comp Neurol 198, 649–665.

ras DP, Vaske T Jr, Hazin FHV, et al. (2009) Stomach contents
Ve
of the pelagic stingray (Pteroplatytrygon violacea) (Elasmo-
branchii: Dasyatidae) from the tropical atlantic. Braz J Ocea-
nogr 57, 339–343.
Von Bonde C (1933) Contributions to the morphology of the
elasmobranchii. J Comp Neurol 58, 377–403.
Von Lu € dinghausen M, Miura M, Wu € rzler N (1999) Variations
and anomalies of the human orbital muscles. Surg Radiol Anat
21, 69–76.
Walker WF, Homberger DG (1992) Vertebrate Dissection, 8th
edn. Philadelphia,PA: Saunders College Publishing.
Wilga CD, Lauder GV (2004) Biomechanics of locomotion in
sharks, rays, and chimeras. In: Biology of Sharks and their Rel-
atives. (eds Carrier JC, Musick JA, Heithaus MR), pp 139–164.
New York,NY: CRC Press.
Williamson M, Keast A (1988) Retinal structure relative to feed-
ing in the rocks bass (Ambloplites rupestris) and bluegill
(Lepomis macrochirus). Can J Zool 66, 2840–2846.
Young GC (2008) Number and arrangement of extraocular mus-
cles in primitive gnathostomes: evidence from extinct placo-
derm fishes. Biol Lett 4, 110–114.
© 2016 Anatomical Society