During their evolutionary history, obligate diurnal lizards lost the classical vertebrate duplex retina containing both rods and cones [Walls, 1942]. Rod photoreceptors were replaced in favor of single and double cones, an adaptation for the demands of a brightly lit environment. Little is known of when this change took place, or whether corroborating physiological limitations led to associated specializations of the lizard eye.
Much of our understanding of the lizard visual system has come from research on the Iguania (comprising iguanids, chameleons, and agamid lizards). These lizards possess four functionally distinct visual pigments, and colored oil droplets positioned at the scleral margin of the inner segment of each photoreceptor [Fleishman et al., 1997; Loew et al., 2002; Bowmaker et al., 2005], a specialization shared only by birds and other reptiles. Gekkonid lizards have a suite of visual specializations which accompanied their shift to nocturnal activity [Citron and Pinto, 1973; Röll, 2000, 2001a; Roth et al., 2009]. There are few descriptions, however, of the visual system among other groups of lizards.
In the late 1980s, rod photoreceptors were described using cytology in the Australian sleepy lizard (Tiliqua rugosa), a day-active member of the Scincidae [Braekevelt, 1989]. This finding, in contrast to the “typical” cone-only retina of diurnal lizards, was questioned [Röll, 2001b]. However, immunohistochemical assays have revealed the presence of rhodopsin (the visual pigment of rods) in the retina of the American anolid (Anolis carolinensis) and the chameleon (Chamaeleo chamaeleon) [Mcdevitt et al., 1993; Bennis et al., 2005]. This raises questions about the classification of lizard photoreceptors. Here, we reexamine the photoreceptors present in the sleepy lizard retina.
The sleepy lizard has a region of heightened ganglion cell density within the central retina [New and Bull, 2011]. Elongated horizontally to form a weak visual streak, this specialization is thought to provide a wide field of distinct vision in its exposed environment. We extend these findings, describing the ocular anatomy of the sleepy lizard eye and the density, distribution and types of retinal photoreceptors, which are the first interface with the visual environment. As the sleepy lizard belongs to the basal Scleroglossa clade of lizards [Vidal and Hedges, 2009], under represented in comparative visual anatomy, this study has the potential to offer new insights into the evolution of the lizard visual system.
MATERIALS AND METHODS
Nine adult sleepy lizards (Tiliqua rugosa aspera) (three ♂, three ♀, and three unknown) were collected from Kadina and Bundy Bore Station, South Australia. They were housed individually in outdoor pens (3.5 × 3.0 m) and fed once every 3 days with sliced fruit, vegetables, and tinned cat food. Water was supplied ad libitum. Animal care and experimental procedures were carried out in accordance with guidelines provided by Flinders University of South Australia Animal Welfare Committee in compliance with the Australian Code of Practice for the use of animals for scientific purposes.
Head and Eye Morphology
The length of the head (rostrum to the tip of the most caudal head scale) and snout (rostrum to nasal eye margin), width of the base of the snout and rostrum (at the position of the nasal eye margin and nares, respectively), and the snout-vent length of each lizard were measured in triplicate using vernier calipers to an accuracy of 0.1 mm. These measurements allowed an estimate of cranial limitations on the breadth of the frontal binocular overlap. Ocular morphology was examined in the eyes of one lizard using an Oculus Pentacam (Oculus, Optikgeräte GmbH). Several scans were taken for each eye under dark conditions and mydriatic agents were not used. Pupil size was measured in two lizards individually placed in an observation chamber of known, stable light intensity. Experiments were conducted at luminances of 0.005 and 430 cd/m2, approximating dim moonlight and typical indoor light levels, respectively. After waiting several minutes to allow lizards to adapt to light levels, subject's eyes were filmed using an infrared sensitive digital HD video camera (Sony Handycam HDR-CX550). Video recordings were converted to jpeg files and four representative images were chosen for each lizard at each light level. Pupil size was measured by fitting an ellipse to the edge of the pupil using Adobe Illustrator CS3 software, and mean pupil area was calculated.
Two specimens were dark adapted, euthanized with Nembutal injected intraperitoneally (∼ 2 cc.) and the eyes excised. Two eyes were paraffin embedded for examination of the anatomy of the eyeball and retina, and two eyes plastic embedded for investigation of photoreceptor distribution. Details of the techniques used are described below.
Anatomy of the Eye
Following fixation in 10% buffered formalin for 48 hr, two eyes were dehydrated in graded ethanols, cleared in chloroform, and embedded in histological paraffin wax. Radial sections of 5 μm thickness were deparaffinized, rehydrated, and stained with Hematoxylin (Harris type) for 10 min and Eosin for 1 min. Images of the eyeball and its components were taken using Q-capture software on an Olympus 5 mega pixel micropublisher digital camera attached to an Olympus BX50 microscope.
Two eyes were hemisectioned at the equator and the lens and vitreous carefully removed. Each eyecup was orientated with a dorsal stitch, fixed in 2% paraformaldehyde for 30 min, and post fixed in 1% osmium tetroxide for 1 hr. Eyecups were cut into individual regions, dehydrated, passed through propylene oxide, and embedded in epoxy resin. Sections were tangentially cut (parallel to retinal layers) at a thickness of 1 μm, double labeled with methylene blue and basic fuchsin, and mounted with Depex mounting medium.
The density, distribution, and packing arrangement of retinal photoreceptors were analyzed using ImageJ™ 1.36b software. Photoreceptors were counted within a 40,000 μm2 field at a depth approximately equal to the ellipsoid body, and double cones were counted as a single entity. Cells overlapping the upper or right edges were recorded, but those overlapping the left or bottom borders of sampling fields were excluded from counts. Cell counts were converted to cells/mm2. No corrections were applied for the slight shrinkage that occurred during the embedding process. Ultrathin tangential sections (100 nm) were stained with 1% uranyl acetate and 0.1% lead citrate for closer examination of photoreceptors on a JEOL 1200EX transmission electron microscope.
To investigate the functional characteristics of sleepy lizard photoreceptors, expressed visual opsins were examined using immunohistochemical labeling. Radially cut paraffin sections (perpendicular to retinal layers, see above) were rehydrated in graded alcohols, rinsed in 0.1 M phosphate buffered saline (PBS), and incubated in 10% normal goat serum (NGS) in PBS for 1 hr at 37°C to limit nonspecific binding of antibodies. Preparations were then incubated overnight at 4°C in a 1:200 dilution of anti-rhodopsin (Rho-4d2) and a 1:200 dilution of either rabbit anti-opsin blue or red-green polyclonal antibody in PBS containing 1% NGS. Rho-4D2 binds to the N-terminal part (amino acids 2–39) of bovine rhodopsin [Hicks and Molday, 1986], the photopigment of rods. The Rho-4D2 antibody is a reliable marker of rods in fish [Knight and Raymond, 1990], amphibians [Hicks and Molday, 1986; Bugra et al., 1992], birds (Hicks, unpublished observation), and mammals [Hicks and Molday, 1986; Hicks et al., 1989], and its specificity has been verified in the retina of an anolid lizard by immunoblotting [Mcdevitt et al., 1993]. Preparations were incubated at 37°C for 1 hr in goat anti-rabbit 594 and goat anti-rabbit 488 secondary antibodies to 1:1,000 dilution in PBS containing 1% NGS, and mounted in glycerol gelatin. Omission of the primary antibody gave no apparent immunolabeling. Consecutive serial sections reacted with different antibodies were photographed with a Zeiss LSM 5 Pascal confocal microscope system and Pascal version 4.0 software (Jena, Germany).
The sleepy lizard has a short, broad head tapering to a rotund snout (Fig. 1). Eyes are placed laterally and anteriorly within the skull, and bordered dorsally by a prominent row of scales forming a distinct brow. This cranial morphology and eye placement provides the potential for a wide visual field and up to 25° of frontal binocular overlap. The eye was globular in shape and the pupil round and dynamic, measuring 3.7 mm2 (St. Dev. = 0.06) in area under low-light levels and constricting to 2.6 mm2 (St. Dev. = 0.15) on exposure to bright light (Fig. 2).
The cornea formed a distinct spectacle, was ∼ 270 μm thick and consisted of five layers; epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium (Fig. 3a). The fibrous stroma comprised 75% of the total corneal thickness and was protected by the thick noncellular Bowman's layer and corneal epithelium. The thin, noncellular Descemet's membrane was discernable between the stroma and corneal endothelium (arrow in Fig. 3A).
Oculus Pentacam imaging allowed examination of the anterior chamber of the eye in vivo. The lens was revealed to be distinctly flat and positioned ∼ 900 μm from the posterior corneal surface at the optic axis (Fig. 3B). Although fixation and dehydration resulted in shrinkage and distortion of the lens, its anatomy was well preserved (see inset Fig. 4). The lens was composed of thin concentrically arranged fibers encapsulated by an epithelial layer (Fig. 4), which was equatorially thickened as an annular pad. Zonular fibers stretching from the equator of the eyes to the ciliary body held the lens in place.
The iris bordered the anterior surface of the lens (Fig. 3B). Dark pigment cells enclosed the iridial stroma, with the double-layered inner epithelium more heavily pigmented than the capillary-rich outer layer (Fig. 4). The sphincter pupillae (SM) contained prominent circular muscle fibers and scattered melanin. Radial muscle fibers of the dilator pupillae (DM) followed the anterior margin of the inner epithelium.
The retina was 250 μm thick adjacent the optic disc and tapered slightly peripherally. It had no distinguishable landmarks and it lacked a fovea. The photoreceptor layer contained densely packed, elongated processes embraced by the retinal pigment epithelium (Fig. 5). The outer nuclear layer was 20 μm thick, composed of two rows of rigidly organized receptor perikarya. The first row of cells was lightly stained and ovoid, the second, bordering the thin outer plexiform layer, round and densely stained. The inner nuclear layer was much thicker than its outer counterpart (50 μm), consisting of loosely organized cells up to 12 cells deep in the central retina. Further vitreal, the inner plexiform layer formed a meshwork of neuronal processes connecting the inner nuclear layer with the ganglion cell layer.
The completely avascular retina was bordered by a well-developed choroid containing numerous, broad venous sinuses intermingled within melanin pigmentation. In addition, the conus papillaris, considered to be a nutritive device for the inner retina [Rodieck, 1973], projected toward the centre of the vitreous chamber from the optic disc. The appearance of a histological section of the conus papillaris cut along its length is shown in Fig. 6. Internally, the conus consisted of an extensive array of capillaries and larger blood vessels separated by a matrix of melanocytes and connective tissue. The optic nerve extended from the eyecup at the base of the conus papillaris.
Photoreceptors projected through the external limiting membrane for up to 30 μm in the dark adapted state (Fig. 7A). The inner segment consisted of a nucleus, a large paraboloid, and an ellipsoid body bordered further scleral by an oil droplet. Outer segments were small (5–7 μm) and conical. Pairs of cone cells were observed with their inner segments in broad contiguity though kept separate by their cell membranes (Fig. 7B). The two members of these pairings were morphologically different; one member short and broad containing a prominent paraboloid body and the second member long and narrow with a small or absent paraboloid body but containing an oil droplet. These cells resembled the accessory and principal members, respectively, of double cones [Fleishman et al., 1997]. Oil droplets were pale yellow in color and located in single cones and within the principal member of double cones.
Photoreceptors were ovoid in tangential cross section, and isolated from those around it by processes of pigment epithelium (Fig. 7B). Cell counts integrated from two eyes revealed that photoreceptors were heterogeneously distributed throughout the retina. Photoreceptor density peaked at 76,600 cells/mm2 (Fig. 8) within the central retina (mean = 50,050 ± SE 6,800 cells/mm2), and decreased peripherally to 30,275 ± 4,370 cells/mm2 (unpaired t test; t = 2.45, df = 14, P = 0.028). The ventral hemisphere contained a significantly higher density of photoreceptors than the dorsal retina (t = 4.45, df = 22, P < 0.001) (Fig. 9A), while there was no significant difference in receptor density between the temporal and nasal retina (t = 0.42, df = 22, P = 0.68) (Fig. 9B).
Double cones comprised ∼ 19% of all photoreceptors throughout the retina (Table 1). Their density strongly correlated with single cone abundance (r2 = 0.73; two-tailed test for significance of correlation coefficient, t = 6.835, df = 17, P < 0.005; Fig. 10). Double cones were interspersed between single cones such that two double cones were rarely adjacent to one another (Fig. 7B). A significant population of photoreceptors exhibited strong immunofluorescence for Rho-4D2 (green labeling in Fig. 7C,D). Double labeling with either rabbit anti-opsin medium-long wavelength or short-wavelength-sensitive polyclonal antibodies (red labeling in Fig. 7C,D, respectively) revealed that Rho-4D2 labeled a different population of photoreceptors not recognized by any of the other antibodies.
Table 1. Regional variation in photoreceptor density and double cone percentage throughout the sleepy lizard retina
Receptor density (mm−2)
Double cones (%)
Cell counts were taken at approximately the ellipsoid body of cone photoreceptor cells and double cones were counted as a single entity.
The Australian sleepy lizard occupies stable overlapping home ranges [Bull, 1987; Bull and Freake, 1999] and maintains long-term monogamous relationships [Bull, 1988, 1990] that require accurate interindividual recognition. Although olfactory cues play a major role in social recognition [Bull et al., 1993], visual cues are also thought to be used [Zuri and Bull, 2000a] and play a significant role in food choice [Wohlfeil, 2008], refuge selection [Kerr et al., 2003; Auburn et al., 2009], spatial orientation [Zuri and Bull, 2000b; Freake, 2001], and the detection of approaching threats [Murray and Bull, 2004]. Clearly, the sleepy lizard is a visual animal, and its eyes are adapted for this lifestyle. The eyeball is globular in shape [New and Bull, 2011], and the lens distinctly flat and positioned forward within the eye, creating a relatively shallow anterior segment. This optical configuration favors visual acuity over sensitivity, maximizing focal length and producing a relatively larger retinal image spread over a greater number of photoreceptors [Land and Nilsson, 2001; Hall, 2008]. The iris dilator and sphincter muscles are well developed, enabling changes in pupil aperture. A dynamic pupil facilitates more rapid retinal adaptation, likely aiding visually controlled movements between exposed areas and lower light levels experienced beneath bushes or within burrows.
Paradox of the Rods
The retina of the sleepy lizard contains only cone photoreceptors which could be separated into two distinct classes; single cones and double cones. The cones are characterized by an oil droplet and small conical outer segments, and their morphology is consistent with descriptions from other diurnal lizards [Peterson, 1992; Röll, 2001a; Barbour et al., 2002; Bowmaker et al., 2005]. In contrast to Braekevelt (1989), no morphological evidence of rods was observed. Röll (2001a) noted that Braekevelt's (1989) description of rods—joining closely with the neighboring visual cell at the level of the ellipsoid—suggested he may have erroneously ascribed the accessory member of double cone photoreceptors as rods. This is plausible given that Braekevelt did not identify double cone photoreceptors, and that the accessory member of sleepy lizard double cones, like in other diurnal lizards, lacks an oil droplet. However, Braekevelt described “rods” as narrow and containing no paraboloid body, while the accessory member inner segment was observed here to be broad and containing a prominent paraboloid body.
Although the sleepy lizard retina contains only cone photoreceptors based on morphological criteria, a subset of photoreceptors display immunoreactivity to Rho-4D2, a monoclonal antibody raised against the N-terminal part of rhodopsin. This is not the first evidence of rhodopsin within a pure-cone retina [Mcdevitt et al., 1993]. Kawamura and Yokoyama (1997) identified a gene orthologous to rhodopsin in other vertebrates in the pure-cone retina of an anolid lizard (Anolis carolinensis) (see references therein). This gene encodes the RH1Ac opsin, and is expressed in addition to the SWS1Ac, SWS2Ac, RH2Ac, and LWSAc visual opsins, and labeled by the Rho-4D2 antibody [Mcdevitt et al., 1993]. Rhodopsin labeling has also been demonstrated in the rod-free retina of the chameleon (Chamaeleo chamaeleon) [Bennis et al., 2005]. Chamaeleo and Anolis both belong to the suborder Iguania [Vidal and Hedges, 2009]. The presence of rhodopsin in a representative species of the Scincidae, which belong to the more basal suborder Scleroglossa, suggests occurrence of the RH1Ac opsin represents the ancestral pattern.
Rhodopsin may enable photoreceptors to lower their threshold to light levels well below that required to stimulate cones, thus affording vision in lower conditions. Cones, are often defined according to the amino acid sequence of their photopigments and in turn the spectral wavelength in which pigments are most sensitive. The spectral and biochemical characteristics of lizard retinal visual pigments are not well understood, and have not been examined at all in the skinks, though lizards appear tetrachromatic. In those diurnal lizards examined, four functionally distinct retinal pigments have been characterized as follows; ultraviolet-sensitive (maximum absorbance λmax range = 365–385 nm; opsin protein = SWS1Ac), short-wavelength-sensitive (440–455 nm; SWS2Ac), medium-wavelength-sensitive (480–505 nm; RH2Ac), and long-wavelength-sensitive (555–625 nm; LWSAc) [Kawamura and Yokoyama, 1997, 1998; Loew et al., 2002; Fleishman et al., 2011]. The discrepancy in the number of opsin genes and functionally distinct pigments is curious. Where is the RH1Ac opsin? Sleepy lizard photoreceptors immunoreactive to Rho-4D2 did not co-label with either the short wavelength or medium-long wavelength opsin antibodies, which likely targeted the SWS1Ac and LWSAc opsins, respectively. The RH1Ac opsin is thus not expressed within these photoreceptors. It has been posited that the RH1Ac pigment is expressed in a medium-wavelength-sensitive photoreceptor together with the RH2Ac opsin pigment [Kawamura and Yokoyama, 1998], but further examination is required. Moreover, cone-like sensitivity to hydroxylamine [Kawamura and Yokoyama, 1998] suggests that the RH1Ac pigment may even be cone-like in sensitivity. Characterization of receptor phototransduction and regeneration rates [e.g., Pugh and Lamb, 2000; Lamb and Pugh, 2006], including among the skinks, will provide a clearer understanding of the functional and adaptive properties of lizard vision.
Photoreceptor Density and Ecology
Specimens were wild caught sexually mature adults, though their exact age was unknown. Age-related differences in photoreceptor numbers have not been reported among lizards, but such differences cannot be discounted as sleepy lizards are long lived [Bull, 1995] and inhabit brightly lit environments. We provide here an assessment of retinal photoreceptors based on two retinas. Although not permitting the description of retinal specializations afforded by topographical retinal maps, it does allow us to examine regional differences in receptor density and their correspondence with the topographic distribution of ganglion cells in this species [New and Bull, 2011].
Photoreceptors are heterogeneously distributed throughout the sleepy lizard retina, peaking centrally at 76,000 cells/mm2 and decreasing peripherally. This is substantially lower than maximum densities observed within foveal specializations, such as the 290,000 photoreceptors/mm2 reported for the central fovea of Anolis carolinensis [Makaretz and Levine, 1980]. That species actively hunts and pursues fast moving insect prey. In contrast, the sleepy lizard moves slowly through its habitat, foraging opportunistically and predominantly on plant matter. This lifestyle would place less demand on a foveal specialization.
Photoreceptor density in Anolis carolinensis drops to 3,200 cells/mm2 adjacent the fovea and to 1,600 receptors/mm2 at the retinal periphery [Makaretz and Levine, 1980], and thus must rely on head and eye movements to center objects of interest onto their central fovea for distinct vision. In contrast, the lowest photoreceptor density we observed in the sleepy lizard eye is an order of magnitude higher than in Anolis carolinensis, 15,900 cells/mm2. The more uniform distribution of photoreceptors in the sleepy lizard retina, together with the nasolateral position of the eyes within the skull, affords a wide field of distinct vision without the need for directed eye movements. The sleepy lizard occupies fully terrestrial open environments, where much of the biologically relevant visual cues, including predators, will come from above. The significantly higher density of photoreceptors within the ventral retina, in correspondence with the topographic distribution of ganglion cells [New and Bull, 2011], supports greater resolving power and sensitivity in the dorsal visual field into which the majority of visual targets might appear.
The sleepy lizard (Tiliqua rugosa) has several visual specializations related to the demands of its diurnal lifestyle in an exposed environment. The eyes are laterally placed and a relatively high density of photoreceptors within the peripheral retina affords a wide field of distinct vision. A large focal length provides a wide image throw onto the retina, and the dynamic pupil enables adjustment of retinal illumination assisting photoreceptor adaptation. Although all retinal photoreceptors contain an oil droplet, a subset of cones also contains rhodopsin. Our results provide further evidence that the traditional categorization of vertebrate photoreceptors into two distinct “morphs” possessing exclusive functional and optical properties is too simplistich.
We are grateful to Michelle Lewis, Kerry Gascoigne, Bill Stell, and Krisztina Valter for providing technical assistance. Thank you also to Dale Burzacott for measurement of sleepy lizard head morphology and Robert Molday for his kind gift of Rho-4D2 antibody used here in immunohistochemistry. Comments from Jochen Zeil and three anonymous reviewers on earlier versions of this manuscript were greatly appreciated.