Morphology of the cornea and iris in the Australian lungfish Neoceratodus forsteri (Krefft 1870) (Dipnoi): Functional and evolutionary perspectives of transitioning from an aquatic to a terrestrial environment

The Australian lungfish, Neoceratodus forsteri (Krefft 1870), is the sole extant member of the Ceratodontidae within the Dipnoi, a small order of sarcopterygian (lobe‐finned) fishes, that is thought to be the earliest branching species of extant lungfishes, having changed little over the last 100 million years. To extend studies on anatomical adaptations associated with the fish–tetrapod transition, the ultrastructure of the cornea and iris is investigated using light and electron (transmission and scanning) microscopy to investigate structure–function relationships and compare these to other vertebrate corneas (other fishes and tetrapods). In contrast to previous studies, the cornea is found to have only three main components, comprising an epithelium with its basement membrane, a stroma with a Bowman's layer and an endothelium, and is not split into a dermal (secondary) spectacle and a scleral cornea. The epithelial cells are large, relatively low in density and similar to many species of non‐aquatic tetrapods and uniquely possess numerous surface canals that contain and release mucous granules onto the corneal surface to avoid desiccation. A Bowman's layer is present and, in association with extensive branching and anastomosing of the collagen fibrils, may be an adaptation for the inhibition of swelling and/or splitting of the stroma during its amphibious lifestyle. The dorsal region of the stroma possesses aggregations of pigment granules that act as a yellow, short wavelength‐absorbing filter during bright light conditions. Desçemet's membrane is absent and replaced by an incomplete basement membrane overlying a monocellular endothelium. The iris is pigmented, well‐developed, vascularised and contractile containing reflective crystals anteriorly. Based upon its ultrastructure and functional adaptations, the cornea of N. forsteri is more similar to amphibians than to other bony fishes and is well‐adapted for an amphibious lifestyle.


| INTRODUCTION
The Dipnoan order of lungfishes are members of the bony fishes (Osteichthyes) and arose during the Devonian period 410 million years ago (Moy-Thomas & Miles, 1971); all three living genera, for example, Neoceratodus with one species from Australia, Lepidosiren with one species from South America and Protopterus with four species from Africa, have fossil records dating back to the Cretaceous era (Broin et al., 1974;Kemp et al., 2017;Kemp & Molnar, 1981;Marshall, 1986;Sige, 1968).Lungfishes (Ceratodontiformes) are all believed to represent the closest living relatives to terrestrial vertebrates or tetrapods (Brinkmann et al., 2004;Tohyama et al., 2000;Venkatesh et al., 2001;Yokobori et al., 1994).Compared with the other species of lungfishes, which have paired lungs, the Australian lungfish Neoceratodus forsteri (Krefft 1870) has a single lung and uses predominantly gill respiration, only breathing air during periods of increased activity or when water quality is poor (Kemp, 1986).Based on these traits and other morphological, molecular and palaeontological evidence across the three genera of lobe-finned fishes, N. forsteri, the sole extant species representing the family Ceratodontidae within the order Dipnoi, is considered to be the earliest branching lungfish having changed little over the last 100 million years (Kemp & Molnar, 1981;Tokita et al., 2005).Based on the importance of this critical stage in vertebrate evolution, studies on the morphology of the Australian lungfish remain a critical approach in tracing adaptations associated with the fish-tetrapod transition (Clement et al., 2022).
With respect to their sensory systems, N. forsteri is thought to rely heavily on non-visual senses such as electroreception (Watt et al., 1999), olfaction and lateral line (Bartsch, 1993) for locating food from the benthos in rivers and streams, where it is relatively turbid, with vision playing a secondary role given their small eye size (Schwab et al., 2006).N. forsteri feeds on molluscs, plants and crustaceans (Joss & Joss, 1995;Kemp & Molnar, 1981), where prey is taken primarily during foraging, achieved by moving their snout from side to side across the substrate (Watt et al., 1999), where the benthos-water interface would be a predominant feature of their visual environment.However, these heavy-bodied, powerful and aggressive omnivores do spend considerable time in slow-moving pools, where clearer freshwater would allow better visibility and the eyes used for visually targeting food and navigating submerged objects.N. forsteri also regularly rises to the river surface and breathes air in times of increased activity, such as during mating or when the water becomes stagnant, thereby periodically exposing the eyes to bright sunlight.
Recent studies of the visual system of N. forsteri have concentrated on retinal photoreception (Bailes, Davies, et al., 2007;Bailes, Robinson, et al., 2006), optics, levels of accommodation and pupillary movement (Bailes, Trezise, et al., 2007), spatial resolving power (Bailes., Trezise, et al., 2006) and visual ecology (Hart et al., 2008;Marshall et al., 2011).These detailed studies reveal that short wavelength light is absorbed by the cornea and lens and focussed onto the retina that contains a heterogeneous distribution of five types of large retinal photoreceptors (one type of rod up to 21 µm in diameter and four types of cone up to 14 µm in diameter) each expressing a different visual pigment (rh1 in the rod and rh2, lws, sws1 and sws2 in the four types of cone), providing the retinal machinery for tetrachromatic vision (at least in juveniles; Bailes, Davies, et al., 2007;Bailes, Robinson, et al., 2006).Hart et al. (2008) assessed the spectral absorption characteristics of the ocular media, the visual pigments and spectral filters (pigmented oil droplets and diffuse pigment within the ellipsoid and paraboloid regions) of the large retinal photoreceptors, the spectral distribution of the ambient light in this species' underwater habitat and the spectral reflectance of relevant objects in its natural environment and established that N.
forsteri is able to enhance its ability to discriminate objects underwater (i.e., plants, logs and conspecifics) on the basis of colour.
Although the eyes of N. forsteri have relatively low spatial resolving power (1.6-1.9 cycles/deg) based on the spacing of their large ganglion cells, they have retained the ability to resolve objects, especially when they venture into more shallow, clearer waters (Bailes, Trezise, et al., 2006), while balancing the trade-off between resolving power and photic sensitivity (Marshall et al., 2011).
According to Appudurai et al. (2016), colour discrimination in South American (L.paradoxa) and spotted African (P.dolloi) lungfishes may be different from N. forsteri, where there are species-specific differences in the complement of retinal photoreceptors (one rod and two cone types in P. dolloi and one rod and one type of cone in L.

paradoxa).
There are only a few previous studies on the structure and contractibility of the iris in lungfishes.Pupillary constriction in most vertebrates is the result of the contraction of the sphincter pupillae muscle within the iris (Walls, 1942).However, Rochon-Duvigneaud (1943) reported the absence of a sphincter pupillae muscle in the lepidosirenid iris, and although Walls (1942) considered that the contractility of epithelial cells of the pars iridica is thought to be responsible for pupillary movement in Protopterus spp., there are no descriptions of the structure of the iris in this species.In N. forsteri, the relative pupil size constricts by an average of 37% after 45 min of exposure to white light after dark adaptation (Bailes, Trezise, et al., 2007).A slight concentric aphakic gap (where the pupillary aperture is larger than the lens diameter) exists at full pupil dilation, while the constricted pupil covers a thin (0.16 mm wide) rim of the lens periphery or 34% of the lens cross-sectional area (Bailes, Trezise, et al., 2007).The pupil of the African lungfish Protopterus annectens was observed to constrict more than 100 years ago (Sleinach, 1890), but, unlike in N. forsteri, the pupil of P. annectens constricts unevenly to resemble a horizontal slit upon light exposure when emerging from an aestivation cocoon, a physiological adaptation to drought that does not occur in N. forsteri.Trends in the magnitude and speed of the pupillary response can be seen between different vertebrate classes, and in this regard, the iris of N. forsteri shares more similarities with amphibians than teleost fishes (Bailes, Trezise, et al., 2007).
The transition from water to land was a key event in the evolution of vertebrates that occurred over a period of 15-20 million years towards the end of the Devonian.Tetrapods, which are phylogenetically nested within sarcopterygians, evolved adaptations for an amphibious existence (Friedman, 1969;Lu et al., 2016;Zhu & Yu, 2002).According to Hart et al. (2008) and based on the diversity of retinal adaptations, the visual system of N. forsteri is more closely aligned to that of modern amphibians and other terrestrial animals than to teleosts and other sarcopterygian fishes (Marshall et al., 2011).
This study is focussed on an examination of the structure-function relationships of the cornea of N. forsteri and whether it more closely resembles that of tetrapods or teleosts.Unfortunately, there are almost no ultrastructural studies of the cornea in lungfishes, with the exception of the surface of the corneal epithelium (H.B. Collin & S. P. Collin, 2000; S. P. Collin & H. B. Collin, 2006).According to Walls (1942), lungfishes have a secondary spectacle (a transparent goggle, which is continuous with the dermis and often described as a dermal cornea) and a scleral cornea (continuous with the scleral eyecup), which are 'apparently separate or at least very separable'.This statement is based on light microscopic observations of the cornea in two species, namely Lepidosiren sp. and Protopterus aethiopicus.This contention has been echoed by Rochon-Duvigneaud (1943), Duke-Elder (1958) andTripathi (1974), who also state that the spectacle and scleral corneas are separated by a thin layer permitting the eye to rotate freely beneath the spectacle.In the African lungfish Protopterus sp., this layer is thought to consist of fine strands of mucoid tissue (Friedman, 1969).Bailes, Trezise et al. (2007) also claim that the cornea is comprised of two layers, the outermost of which is distinctly yellow in both juveniles and adults, but these authors provide no structural evidence for the differentiation of these two corneal components.
Here, we describe the functional anatomy of the cornea in the Australian lungfish, N. forsteri and reveal it possesses only one stroma comprised of regular collagenous lamellae and representing the majority of the thickness of the cornea (without a separate spectacle), with no ability for the globe to move or rotate beneath the 'spectacle' to which it appears to be firmly attached (see reviews by Collin & Collin (2001) and Webb et al. (2019) for the general structure and adaptations of the vertebrate eye including the cornea).We also make comparisons of this species' corneal adaptations with the cornea of a range of fishes and tetrapods to assist in our understanding of the evolution of the vertebrate cornea and its ability to meet the demands of clear vision across a range of environmental conditions, for example, in water and air.Although All animals were killed in accordance with the ethical guidelines of both the National Health and Medical Research Council of Australia and the University of Queensland; they were killed with an overdose >20 mL/L of benzocaine (Sigma-Aldrich Inc.) dissolved in acetone (50 g/L), and then the spinal cord was sectioned (Animal Ethics Committee Permit No. ANAT04360040ARC).All animals appeared in good health before euthanasia and none showed evidence of ocular disease or abnormality.Following euthanasia, the eyes were enucleated and the corneas dissected free and fixed in Karnovsky's fixative (2% paraformaldehyde, 2.5% glutaraldehyde, 0.1 M sodium cacodylate buffer, 2% sucrose and 0.1% calcium chloride, pH 7.2) and rinsed in two changes of 0.1 M sodium cacodylate buffer.All corneas were postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer, followed by dehydration in a graded series of alcohols.Both central and peripheral regions of the cornea were examined in all individuals.

| Scanning electron microscopy (SEM)
Corneas from two individuals (left and right eyes) were critically point-dried in a Polaron critical point dryer and mounted on 10 mm aluminium stubs with double-sided tape.Each cornea was hemisected so that half of the cornea was inverted and both sides (epithelial and endothelial surfaces) were displayed.The mounted specimens were coated with 12-15 nm of gold-palladium in a Polaron sputter coater and placed in an oven at 40°C overnight BARRY COLLIN ET AL.
| 3 of 24 before being examined.The epithelial and endothelial corneal surfaces were examined using a Jeol field emission scanning electron microscope with an accelerating voltage of 3 kV.Results were recorded both on 35 mm film and digitally.The areas of individual epithelial cells (in µm 2 ) were obtained by digital analysis of the computer images using NIH Image.The number of cells measured for each species varied from 30 to 100 cells.Using this procedure, the mean epithelial cell density and standard deviation were calculated, providing the extent of variation in cell area (polymegathism).
Interspecific cell densities were compared using either one-or twotailed t-tests for independent variables.Measurements of microvilli, microholes and other surface features were performed on photographic prints or digital images using a magnifier and graticule and calibration tool in Adobe Photoshop, respectively.At least 20 examples of each surface feature were measured (±SD).The central and peripheral regions of the cornea were analysed in all species and both left and right eyes were compared.Since Doughty (1990) reports a tissue contraction of 35.8 ± 1.2% as a result of fixation and critical point drying of rabbit cornea, a correction factor should be applied to the data presented to give an estimate of the in vivo cell dimensions and density.It is also possible that corneal shrinkage differs across taxa, but no attempt was made to assess the degree of species-specific shrinkage in this study.

| Transmission electron microscopy (TEM)
For TEM, corneas were fixed in Karnovsky's fixative (2.5% glutaraldehyde, 2% paraformaldehyde, 2% sucrose in 0.1 M cacodylate buffer, pH 7.2) and postfixed in 1%-2% osmium tetroxide.Tissue was then embedded in araldite or Spurr's low-viscosity embedding media and semithin and ultrathin sections were cut on an ultramicrotome (LKB Ultrascan XL) in transverse and tangential sections.Selected semithin sections were stained with Toluidine blue for examination using a BH-2 Olympus compound light microscope and photographed on an Olympus DP-30 digital camera fitted with a trinocular C mount.Ultrathin sections (70-90 nm) were cut using a diamond knife (DiATOME 45°), collected on copper grids with 200 mesh or rectangular 75/300 mesh (ProSciTech) and stained using lead citrate (Reynolds, 1963) and uranyl acetate according to Collin et al. (1999).
Grids were examined using a Philips 410 TEM and photographed using Kodak Technical Pan black and white film rated at 100 ASA or a Jeol JEM-2100 LaB6 TEM operated at 80 kV and photographed on a Gatan Orius SC 200 CCD camera.

| Cryosectioning
The left eye of an adult lungfish was enucleated from its orbit following euthanasia and the unfixed eye was frozen immediately in liquid nitrogen (Sakura Fine Technical Co., Ltd.) and embedded in optimal cutting compound.Axial sections were cut every 20 µm on a Leica CM3050S cryostat (Leica Microsystems AG) and the remaining block face was photographed using a Sony Cybershot DSC-F828 digital camera (Sony Corp.) fitted with a macro lens.

| Quantitative analyses
Between 20 and 50 examples of each corneal feature were measured (±SD) and dimensions were compared using a two-tailed t-test for independent variables.Measurements were performed on both left (n = 3) and right (n = 3) eyes of six individuals, but all features were not found to be significantly different for size-matched individuals (the eyes in N. forsteri continue to grow throughout life; Bailes, Trezise, et al., 2007) and in left and right eyes, as has been found for corneal features in a range of other vertebrate eyes (Nam et al., 2006;Werther et al., 2017;S. P. Collin & Collin, 2021a).Therefore, the data for left and right eyes were pooled.Due to the difficulty in differentiating juvenile and adult lungfishes, ranges are provided for each corneal dimension.

| General features of the eyes, cornea and iris
The eyes of the Australian lungfish, N. forsteri are laterally placed and protrude beyond the contour of the head (Figure 1a).The cornea appears as a transparent covering of the eye, which becomes thicker at the limbus, where it appears as an opaque border (Figure 1b).The iris is mottled brown in colour, interrupted by irideal regions of yellow/golden reflective material, which appears concentrated as a rim around the pupil margin (Figure 1b).The conjunctiva is dark brown before giving rise to a dorsal arc of lightly pigmented skin dorsally.In the transverse (frozen) section of the unfixed eye, the dorsal region of the cornea is coloured yellow and is continuous with the skin surrounding the eye (Figure 1c).The margins of the circular iris do not extend beyond the diameter of the near-spherical lens in the constricted state but enclose a narrow aphakic space when the pupil is fully dilated.
The cornea of N. forsteri differs from many other members of the bony fishes, in that it has only three main components: an epithelium with its basement membrane, a stroma with a Bowman's layer and an endothelium (Figure 1d).Consistent with the range of body size and maturity of the specimens of N. forsteri studied, the thickness of the cornea ranged from around 130 to 282 µm in the centre with a significant increase in corneal thickness in the periphery (from 191 to 406 µm).

| Corneal epithelium
The epithelial surface is composed of mostly straight-sided pentagonal and some hexagonal cells (Figure 2a) with a previously published cell density of 7843 ± 2214 cells/mm 2 (Collin & Collin, 2006; Table 1).
The surface of these cells is covered by numerous microvilli (Figure 2b), which are 328 ± 131.3 nm in length and 124.6 ± 25.9 nm in width.The width of these microvilli is slightly less than previously published (150 ± 17 nm; Collin & Collin, 2006).Some holes were seen in the surface and these measured 450.4 ± 72.5 nm in diameter, which is greater than found previously (304 ± 82 nm; Collin & Collin, 2006), indicating that there may be both regional and sizerelated differences.Using both SEM and TEM, the thickness of the epithelium of the cornea was 33.8 ± 4.4 µm, with only a slight increase in the periphery.
There are three to four layers of non-keratinised, stratified cells, the arrangement and shape of which appear to fit the criteria for scutoids as presented by Gómez-Gálvez et al. (2018).Scutoids are considered geometrical solutions to the three-dimensional packing of epithelial cells to achieve stabilised bending.The superficial cells have a straight superficial border with numerous columnar structures opening onto T A B L E 1 Summary of the cell density and presence (P) or absence of type(s) of microprojections of the epithelial cells of the anterior surface of the cornea across different classes of vertebrates.The intermediate cells of the corneal epithelium are rather flattened and contain numerous mucous vesicles (Figure 3a-c).
cells are attached to each other with large desmosomes up to 400 nm in length (Figure 3c).The basal epithelial cells in the centre of the cornea are broad (15.21 ± 3.28 µm in width) and squat (10.65 ± 2.15 µm in height) (Figure 3a) compared with those in the periphery, which are narrower (8.92 ± 2.27 µm) and taller (22.05 ± 4.63 µm).
Below the epithelium is a well-formed basement membrane with a thickness of 288.5 ± 104.2 nm.However, apparently randomly scattered throughout the cornea are numerous extensions of the basement membrane toward the epithelium, to a total height of 678.9 ± 242.7 nm (Figure 3d), which appear to contain protrusions of collagen fibrils of the corneal stroma.The basement membrane is attached to the epithelium by numerous hemidesmosomes and to the stroma via anchoring fibrils (854.0 ± 212.8 nm in length), which terminate in anchoring plaques with a diameter of 73.04 ± 16.75 nm.

| Corneal stroma
The epithelium and anterior portion of the stroma in N. forsteri are continuous with the skin surrounding the eye, while the posterior portion of the stroma is continuous with the sclera supporting the posterior of the eye.The stroma has a thickness ranging from 90 to 195 µm in the centre and from 131 to 510 µm in the periphery.The peripheral thickness of the stroma is the major contributor to the peripheral thickening of the cornea (Figure 1c,d).
The corneal stroma consists of approximately 50 lamellae of collagen fibrils.The fibrils in each lamella are parallel and are approximately at right angles to those of the lamellae on either side (Figure 4a).The collagen fibrils have a thickness of 34.2 ± 3.8 nm in the anterior stroma but are significantly (p < .00001)thinner (22.4 ± 2.6 nm) in the posterior stroma.Both groups of collagen fibrils have a similar D-periodicity of 51.5 ± 5.3 nm.In the most superficial (anterior) region of the stroma, the collagen fibrils are randomly arranged and constitute a Bowman's layer as has been found in some other species of vertebrates (Table 2), with a thickness of 2.04 ± 1.14 µm (Figure 3d).The anterior collagen lamellae are very narrow compared to the remaining (more posterior) lamellae and possess marked levels of branching and anastomosing of the collagen fibrils (Figure 4).These branches may consist of one or two collagen fibrils or large bundles of fibrils (Figure 4b-e).Although keratocytes occasionally cross from one lamella to another (Figures 4e and 5), no vertical sutures are present.
Keratocytes are scattered throughout the stroma in all layers: anterior, middle and posterior.They are long and thin and are mostly situated between the lamellae (Figure 5a).Many contain large amounts of glycogen granules (Figure 5a,b).Scattered among the anterior lamellae within the dorsal region of the cornea are flattened cells containing pigment granules, each granule with a mean diameter of 733.2 ± 16.75 nm (Figure 5c).

| Corneal endothelium
Posterior to the stroma is a monocellular layer (endothelium) with a mean thickness of 308 ± 141 nm.The surface of the endothelium is predominantly smooth, but there are occasional extensions of the cell membrane into the anterior chamber (Figure 6a).The junctions between endothelial cells are characterised by the interdigitations of each opposing cell as seen using both SEM (Figure 6a) and TEM (Figure 6b,c).On the stromal side of the endothelium is an irregular and incomplete basement membrane with a thickness of 141 ± 66 nm (Figure 6c).This basement membrane in no way resembles a Desçemet's membrane, which is absent in this species.There is no basement membrane on the anterior chamber aspect of the endothelium or the innermost surface of the cornea opposing the aqueous humour.Other features associated with the endothelium found in the cornea of some other species of bony fishes (Osteichthyes), such as an autochthonous layer, an iridescent layer or an annular ligament (Collin & Collin, 2001) are not present in N.

| The iris
The iris of N. forsteri consists of an anterior stroma and two layers of were not observed despite a previous finding of a small, but significant, constriction of the pupil in response to exposure to white light (Bailes, Trezise, et al., 2007).However, no markers (i.e., smooth muscle-specific actinin) were employed, so further work is necessary to elucidate this finding.Deeper in the stroma lie numerous large, rounded cells (up to 16 µm in diameter) containing large, closely packed pigment granules up to 2.2 µm in diameter (Figure 7c).
The anterior pigment epithelium has a mean thickness of 8.34 ± 3.66 µm and contains oval-shaped pigment granules measuring 918.9 ± 232.8 nm in their long axis and 376.0 ± 88.2 nm in width.An unusual feature is the presence of a basement membrane (47.7 ± 11.1 nm thick) on the anterior (stromal) surface of the anterior pigment epithelium that extends into a complex of finger-like protrusions, with a separation of 76.3 ± 11.8 nm from the cell membrane (Figure 8c).The pigment granules within the anterior pigment epithelium, when viewed in tangential section, appear to contain linear extensions (Figure 7f).The posterior pigment epithelium is thicker (11.76 ± 4.51 µm) with large nuclei and round pigment granules (933.3 ± 257.1 nm in diameter; Figures 6e, 7b and 8a), although, in the extreme periphery, the pigment epithelium thickens and becomes pigment-free.In the extreme periphery of the cornea and in the iris, in association with the blood vessels, there are cells with lobular nuclei, containing numerous granules, some of which contain membranous structures, indicating that they may be producing various mediators or hormones (Figure 8a-d).

| DISCUSSION
The structure of the cornea and iris of the Australian lungfish N.
forsteri is described for the first time and is found to possess features in common with many tetrapods and adaptations to its amphibious freshwater environment.

| Epithelial cell density and surface microprojections
The  Collin, 2000).Marine vertebrates require more active transport of salts and water out of the cornea to maintain an appropriate level of stromal dehydration, while a relatively low epithelial cell density is found in species that frequent estuarine and freshwater conditions such as N. forsteri.Low epithelial cell densities, that is, larger epithelial cells, persist in the corneas of terrestrial vertebrates, which assist in the removal of water from the cornea and the maintenance of corneal transparency (Collin & Collin, 2000).
In air and when the non-aestivating N. forsteri ventures onto land, evaporation from the tears increases the osmolarity of the corneal tear film, thus aiding deturgescence or state of relative dehydration necessary to maintain the transparency of the cornea, which is mostly controlled by the corneal endothelium (Collin & Collin, 2000).
The corneal epithelial surface of N. forsteri has a covering of dense  Collin, 2006;Simmich et al., 2012; Table 1).Microridges are the least effective structures for increasing the cell surface area (vital for assisting the transport of nutrients and oxygen to the cornea) and are mainly found in species occupying a marine environment (Simmich et al., 2012), whereas microvilli, with relatively higher surface area for exchange, appear to be essential for species from terrestrial environments (Beuerman & Pedroza, 1996).Microvilli are predominant in the majority of terrestrial species that have been examined, including many species of birds, amphibians, reptiles and mammals with the exception of the koala T A B L E 2 (Continued) The differences in the findings for Bowman's membrane in the mouse, dog, rat and rabbit may be due to a lack of an appropriate definition. c The basement membrane refers to the thin membrane on the stromal side of the corneal endothelium, which is sometimes present as an alternative to a well-developed Desçemet's membrane, in which banding is present.In the Clearnose skate, in addition to the absence of Desçemet's membrane, there is also no endothelium.Phascolarctidae cibereus, which possesses both microplicae and microridges (Collin & Collin, 2000, Table 1).Two species of deep-sea chondrichthyans, namely, the black shark Dalatias licha and the ratfish Hydrolagus colliei possess microvilli (Collin & Collin, 2000), which may increase the surface area for metabolic exchange within an environment that is relatively still and almost devoid of hydrodynamic movement (Table 1).

| Epithelial surface canals and the epithelial basement membrane
The presence of cylindrical canals (underlying microholes) in the corneal epithelial surface of N. forsteri, which contain and release mucous granules onto the corneal surface, appears to be unique among the bony fishes.They have been described in the dermal cornea of two species of lampreys, that is, the pouched lamprey Geotria australis (Collin et al., 2021) and the shorthead lamprey Mordacia mordax (Collin et al., 2023), in the ammocoete larva of the sea lamprey Petromyzon marinus (Dickson et al., 1982) but not in the adult (Dickson et al., 1982;Van Horn et al., 1969) and are present, although less common and less well-developed, in the cornea of the postmetamorphic axolotl Ambystoma mexicanum (Collin & Collin, 2021a).They have not been reported in any teleosts.
The corneal epithelial canals in N. forsteri do not resemble the goblet cells found in the corneal epithelium of some teleosts, including the salamanderfish Lepidogalaxias salamandroides (Collin & Collin, 1996), but are channels designed to allow the mucous vesicles, produced in the cytoplasm of the intermediate cells of the epithelium to be expelled onto the corneal surface to protect it against dehydration, when exposed to air and damage associated with burrowing.In addition to these species with cylindrical surface canals, holes on the surface of the corneal epithelial cells have been described in the black shark D. licha (Chondrichthyes) (Collin & Collin, 2006), however, whether these are associated with similar canals or with the release of mucus is unknown.
Elevations of the basement membrane into folds in the corneal epithelial cell membrane in N. forsteri are also found in the little penguin Eudyptula minor (Collin & Collin, 2021b) and the shorthead lamprey M. mordax (Collin et al., 2023).An irregularity of the posterior cell membranes of the basal epithelial cells also occurs in humans (Gipson, 1994;Hayashi et al., 2002), although the basement membrane is not as well developed in humans as in N. forsteri and it does not show the same variations in thickness and is not present within these indentations.

| Bowman's layer
Bowman's layer is a somewhat enigmatic structure in the cornea of numerous species of vertebrates.Its existence throughout the vertebrates, and in particular in the Australian lungfish, N. forsteri and possibly in some species of lampreys (Agnatha), suggests it may be an ancestral characteristic in the evolution of the cornea (Collin & Collin, 1993).In lampreys, Dickson and Graves (1981) found a 'thin layer analogous to Bowman's' in the European river lamprey Lampreta fluviatilus, in contrast to Rochon-Duvigneaud (1943), who claimed it is absent.Similarly, in the sea lamprey P. marinus, Bowman's membrane was claimed to be absent by Rochon-Duvigneaud (1943) but present by Van Horn et al. (1969) and Pederson et al. (1971).A Bowman's layer is not present in the pouched lamprey G. australis (Collin et al., 2021) or the shorthead lamprey M. mordax (Collin et al., 2023).
Both Duke-Elder (1958) and Tripathi (1974) considered Bowman's layer to be a normal component of the teleost cornea, although there are many exceptions.In amphibians, it is present in the cornea of both the pre-and postmetamorphosed axolotl A. mexicanum (Collin & Collin, 2021a) but is not found in the salamander Triturus c.
cristatus (Margaritis et al., 1976), two species of frog, Rana pipiens and Rana catesbeiana (Kaye, 1962) or a tadpole, Rana temporaria ornativentris (Hayashi et al., 2002).According to Walls (1942), Bowman's layer is seldom discernable in mammals and is not discernable in marsupials.Other studies claim it is present in most mammals (Gipson, 1994), including the mouse, rat, guinea pig, cat, rabbit, cattle and humans (Hayashi et al., 2002).However, it has also been reported as absent in the mouse (Whitear, 1960), rat (Jakus, 1956), cat and dog (Kafarnik et al., 2007) and rabbit (Gipson, 1994;Margaritis et al., 1975Margaritis et al., , 1976)).A Bowman's layer has been found in birds (Gipson, 1994;Kafarnik et al., 2007), including the golden eagle Aquila chrysaetos (Murphy & Dubielzig, 1993) and the little penguin E. minor (Collin & Collin, 2021b; Table 2).The differences of opinion regarding the presence of a Bowman's membrane in various species may derive from the range of techniques, such as light microscopy, corneal confocal microscopy and TEM, used to observe the layer and the lack of detailed criteria to define a Bowman's membrane for each of these techniques.
The specific role of Bowman's layer is uncertain, although with the inclusion of hemidesmosomes, a basement membrane and anchoring fibrils, which penetrate the anterior cornea and are more prominent in the presence of a Bowman's membrane, it is clear that it forms part of the mechanism to attach the epithelium to the corneal stroma (Gipson, 1994).At least in teleosts, and especially in marine species, it may be part of the corneal barrier to sodium and water movement (Edelhauser & Siegesmund, 1968).However, it is unable to modulate the passage of moderate-to large-sized proteins, which may indicate a reduction in barrier function (Wilson, 2020).According to Collin and Collin (1998), it may add additional strength and inhibit splitting of the anterior cornea, which, for N. forsteri, may be an important adaptation of the evolution of the cornea during the fish-tetrapod transition (see below).
4.4 | Adaptations of the corneal stroma Walls (1942) claims that the cornea of N. forsteri consists of two layers, an inner (scleral) cornea and an outer dermal cornea or secondary spectacle, an ocular adaptation that is associated with coming out of the water into air and/or of probing for food on a sandy or muddy bottom and 'occurs in practically all amphibious fishes'.He also claims that the fibrous layer of the sclera continues anteriorly to form a portion of the corneal stroma, entirely unconnected with the skin of the dermal spectacle and apparently separate from the anterior component (Walls, 1942).According to Rochon-Duvigneaud (1943), the cornea of the South American lungfish, L. paradoxa, comprises a cutaneous coat, at a level at which the mucous cells have disappeared, and a thinner scleral layer forms the ocular globe.Between the two is a thin layer permitting the globe to slide freely.Similarly, Walls (1942) states that in both, P.
aethiopicus and Lepidosiren sp., the eyeball is able to turn freely under a transparent secondary spectacle with Duke-Elder (1958) also claiming that in Protopterus, the dermal and scleral cornea are separated.In N. forsteri, we found a single cornea with no apparent line of weakness for possible separation of the stroma into dermal and scleral components, even though the anterior portion of the cornea is continuous with the skin and the posterior portion is continuous with the sclera of the globe.Hence, there is no evidence that the eyeball of N. forsteri can rotate freely beneath a dermal cornea or secondary spectacle.
In N. forsteri, there are keratocytes present within all levels of the stroma, both anterior and posterior.If the cornea of N. forsteri has two separable stromal components, this finding is in contrast to several species of teleosts, in which there are both dermal and scleral corneas separated by mucoid or other layers and in which the scleral corneal stroma is devoid of keratocytes, namely, in three species of gadiform fishes, the rattail, Nezumia aequalis, the Pacific tomcod, Microgadus proximus and the armoured grenadier, Coryphanoides (Nematonorus) armatus (Collin & Collin, 1998) and in two species of pipefishes, Syngnathus sp.(Nicol, 1989) and Corythoichthyes paxtoni (Collin & Collin, 1995).Only in the salamanderfish, L. salamandroides, have a few scattered keratocytes been described in the scleral component of a divided cornea (Collin & Collin, 1996).
Although some teleosts have two or even three stromas consisting of lamellae of collagen fibrils and separated by various structures, N. forsteri has only one, and there is nothing visible in its stroma to indicate that it is capable of division.Separate dermal and scleral stromas have been described in the pipefish, C. paxtoni (Collin & Collin, 1995), and the salamanderfish, L. salamandroides (Collin & Collin, 1996), while three species of deep-sea gadiforms possess three stromas: a dermal cornea and two scleral corneas (Collin & Collin, 1998).There is only one stromal component of the cornea in three species of trout (Edelhauser & Siegesmund, 1968), the sandlance, Limnichthyes fasciatus (Collin & Collin, 1988), the Florida garfish, Lepisosteus platyrhincus (Collin & Collin, 1993) and the zebrafish, Danio rerio (Zhao et al., 2006).
The extensive lamellar branching and anastomosing found in the cornea of N. forsteri is present in many species and is claimed to be most common in birds, less common in reptiles, followed by amphibians and least common in fishes (Koudouna et al., 2018;Winkler et al., 2015).See Table 2 for a summary of the presence or absence of branching and anastomosing of collagen lamellae across different classes of vertebrates.
Collagen lamellar branching and anastomosing, along with angled lamellae, greatly influence tissue mechanics and control corneal shape in the mammalian cornea (Koudouna et al., 2018) and presumably also in N. forsteri, and hence help maintain the orthogonal and rotational organisation of the collagen lamellae for transparency under various environmental conditions.
The diameter of the collagen fibrils in the anterior corneal stroma (34.2 nm) of N. forsteri is significantly greater (p < .00001)than in the posterior stroma (22.4 nm).Even though the cornea is not divided, this difference is consistent with the finding that the collagen fibrils of the dermal cornea are thicker than those of the scleral cornea as reported in two teleosts, that is, the Pacific tomcod M. proximus and the armoured grenadier, C. (N.) armatus (Collin & Collin, 1998), and in two species of lampreys, that is, the pouched lamprey, G. australis (Collin et al., 2021), and both the upstream and downstream migrants of the shorthead lamprey, M. mordax (Collin et al., 2023).However, collagen fibril diameter is similar in the deep-sea rattail, N. aequalis (Collin & Collin, 1998).

| Corneal pigmentation
The inclusion of concentrations of pigment granules within flattened cells of the anterior (dorsal) lamellae in N. forsteri suggests that this species contains a method of spectrally filtering light as it enters the eye.This study anatomically confirms earlier reports of yellow pigmentation within the dorsal cornea of both juvenile and adult individuals, where the ocular media (cornea and lens) spectrally filter out wavelengths below 330 and 400 nm in juveniles and adults, respectively (Marshall et al., 2011).Yellow corneas have been described in a number of diurnal fishes (i.e., toadfishes, sculpins and pikes) living in bright light environments and are known to prevent the potentially damaging ultraviolet wavelengths from reaching the delicate retina, reduce chromatic aberration and intraocular glare and improve contrast vision (Heinermann, 1984;Orlov & Gamburtzeva, 1976;Siebeck et al., 2003;Walls & Judd, 1933).In N.
forsteri, the pigment aggregations within the dorsal region of the cornea that is exposed to the brightest incident sunlight when these fishes are at the water's surface, in conjunction with a number of coloured photoreceptor inclusions (Bailes, Robinson, et al., 2006) will undoubtedly reduce retinal damage but also reduce light absorption and photopic sensitivity (Hart et al., 2008;Marshall et al., 2011;Walls & Judd, 1933).The large diameter of the retinal photoreceptors (of both rods and cones) in N. forsteri may therefore compensate for the loss of sensitivity while still providing the ability to chromatically discriminate between the macrophytes on which it feeds (Bailes, Robinson, et al., 2006;Hellström et al., 2011).

| Desçemet's membrane
The basement membrane on the stromal side of the endothelium of N. forsteri does not resemble a Desçemet's membrane, which is absent in this species.Walls (1942) described Desçemet's membrane and mesothelium in P. annectens as the 'thinnest imaginable', although this was before the development of the TEM and the studies of Desçemet's membrane that followed.Desçemet's membrane in humans and most mammals is now known to be welldefined, with evidence of continued growth on the posterior (endothelial) side (Gipson, 1994;de Oliveira & Wilson, 2020), the presence of banding in the anterior portion (100-110 nm in humans; Gipson, 1994;Jakus, 1956;Murphy et al., 1984) and a thickness of 3-12 µm in humans (Gipson, 1994) or from 1 to 20 µm in various species of mammals (Hayashi et al., 2002).
However, Desçemet's membrane is absent and replaced by an irregular and incomplete endothelial basement membrane with a thickness of only 118 ± 49 nm in N. forsteri, and is absent in both the Florida garfish L. platyrhincus (Collin & Collin, 1993) and the carp Cyprinus carpio (Smelser & Chen, 1954).In contrast, in three species of deep-sea gadiform fishes, Desçemet's membrane is described as well-formed, with thicknesses between 100 and 230 nm (Collin & Collin, 1998), although no banding was described so these may not be true Desçemet's membranes.Walls (1942) claims that Desçemet's membrane is thin in all lampreys.However, it has been shown recently that a Desçemet's membrane is absent in both the pouched lamprey, G. australis (Collin et al., 2021) and the shorthead lamprey, M. mordax (Collin et al., 2023) and that it is replaced by very thin basement membranes (86 nm in G. australis and 118 nm in M. mordax) on the stromal side of the posterior monocellular layer and these show none of the characteristics of a true Desçemet's membrane.
In the little penguin E. minor, another amphibious species, Desçemet's membrane is well-developed, relatively thick (0.616-1.471 µm), with apparent banding (109 ± 8 nm) in the anterior portion and evidence of marked growth throughout life in the posterior section (Collin & Collin, 2021b).In addition to anchoring the endothelium, Desçemet's membrane has a crucial role in modulating the entry of nutrients and growth factors, such as tumour growth factor 1, into the corneal stroma, where the presence of myofibroblasts could lead to stromal fibrosis, loss of corneal transparency and reduced vision (de Oliviera & Wilson, 2020).Another function of Desçemet's membrane may be biomechanical to distribute even tension and prevent gross deformation of the cornea after swelling (Collin & Collin, 1993;de Oliviera & Wilson, 2020), with its elastic properties likely to serve an important role in the overall maintenance of the structure and curvature of the entire cornea and its overall refractive power.Its absence in at least three species of lamprey, being replaced by a thin and incomplete basement membrane (Collin et al., 2021(Collin et al., , 2023;;Pederson et al., 1971) and in elasmobranchs, for example, the clearnose skate R. eglanteria (Conrad et al., 1994), may be related to the presence of vertical sutural fibrils in the stroma of these species, which have taken on this role (Collin & Collin, 1993).
The presence of lamellar branching and anastomosing may also play a role in the prevention of corneal swelling, with interconnections between the lamellae minimising the separation between the collagen fibrils associated with hydration, as they are present in the corneas of N. forsteri, and other bony fishes (Table 2), in the absence of both a true Desçemet's membrane and vertical sutures.The sutures described in the gadiforms (Collin & Collin, 1998) are really branching rather than vertical sutures as recently defined (Collin et al., 2021).
An additional feature distinguishing N. forsteri (and Protopterus sp.;Tripathi, 1974) from other fishes is the absence of an annular ligament, which has been described in two species of agnathans, that is, the pouched lamprey G. australis (Collin et al., 2021) and the shorthead lamprey M. mordax (Collin et al., 2023) and some species of teleosts (Table 2).

| Iris
In N. forsteri, the iris is well developed comprising a substantial stroma with an average thickness of 36.51 µm and two prominent pigment cell layers.In contrast, the iris has been described as thin in both, Protopterus sp.(Tripathi, 1974) and Lepidosiren sp.(Walls, 1942).Rochon-Duvigneaud (1943) described the iris in L. paradoxa as uniquely composed of only two layers, that is, an anterior layer of tall (70 µm) cells and a posterior layer of small cells, and either lacking or containing a little developed mesoderm.Rochon-Duvigneaud (1943) claimed that the absence of an iris mesoderm, or its poor development, is the result of a 'developmental fault' and not the result of resorption.This is not consistent with our findings for N. forsteri.
Consistent with our findings, Bailes, Trezise et al. (2007) reported that the iris contains gold-reflecting material, which is mostly obscured by the dark pigment around the pupillary margin.In fact, the clear (presumed guanine) crystals are merely reflecting the light having passed firstly through the cornea, then back through the yellow cornea for a second time to give the golden appearance.
Although the colour of the iris is derived from the iris pigmentation, the size of the pigment granules is not considered to contribute to the colour (Imesch, 1996).Hence, why there is such a broad range of sizes and shapes of melanosomes in the iris of N. forsteri is unknown.
According to Imesch (1996), the number and/or density of the melanosomes in the cytoplasm of the cells may give rise to the colour of the iris rather than the size of the pigment granules.Alternatively, the presence of differently shaped granules, that is, oval pigment granules as seen in N. forsteri, may be the defining factor.In the skin of mice, Hellström et al. (2011) find that a change in the shape of melanosomes to oval or ellipsoid is associated with a change in the colour of the skin.Bailes, Trezise et al. (2007) reported that N. forsteri possesses a mobile pupil with a slow pupillary response.This is consistent with our findings, but we were unable to identify any muscle fibres (sphincter pupillae muscles) in the iris stroma.Similarly, muscle fibres are thought to be absent in P. annectens (Tripathi, 1974;Walls, 1942) and in L. paradoxa, Rochon-Duvigneaud (1943) could find no evidence of an iris sphincter.Further work is needed to identify the source of pupillary movement in lungfishes.
Given the unique phylogenetic position of the Australian lungfish, N.
forsteri, within the sarcopterygian (lobe-finned) fishes and its amphibious lifestyle, the functional morphology of the cornea and iris was investigated to understand how the cornea meets the demands of a water-to-air transition and the origins of the tetrapod eye.The cornea is not divided into dermal and scleral components, as occurs in some amphibious fishes and possesses relatively low non-aestivating, N. forsteri ventures onto land, where there is the risk of corneal (stromal) dehydration, which is critical to maintaining transparency.The morphology of the iris is examined in N. forsteri to confirm or refute early reports of this structure being poorly developed in lungfishes, and what gives rise to its colour and mobility.2 | METHODS 2.1 | Source of animals and preservation of eyes Three juveniles (35-75 cm in TL) and three adults (75-95 cm in TL) of the Australian lungfish N. forsteri (Krefft 1870) were caught with (barbless) hook and line from the Mary River, Queensland (Queensland Fisheries Management Authority Permit No. PRM01599G).All animals were transported to the University of Queensland and maintained in freshwater aquaria under a 12:12 light/dark cycle and fed a diet of commercial fish food.While maintained in aquaria, the head and eyes of the lungfish were photographed using a Nikon digital camera (D5600).Some previously preserved ocular and corneal tissue was also donated by J. Joss (following approval by the Macquarie University Animal Ethics Committee Permit No. 2006/ 015) and N. A. Locket (tissue also provided by J. Joss but consequently embedded in Spurr's resin and sent to S. P. Collin, see below).

F
I G U R E 1 Neoceratodus forsteri, eyes and cornea.(a) Frontal view of a juvenile showing the head and lateral position of the eyes.(b) Close-up of the eye.Note the brown-mottled appearance of the iris with regions of golden reflectivity including around the rim of the circular pupil.The asterisk depicts the thickening of the cornea in the limbic region.(c) Axial section of the freshly frozen adult eye showing the near-spherical lens (l), iris (i) and cornea (arrows).The dorsal region of the cornea (to the left) contains yellow pigmentation.Arrowheads indicate the retina that lines the eyecup and the asterisk signifies an increase in corneal thickness.(d) Low-power electron micrograph showing the three main components of the lungfish cornea: epithelium (e), stroma (s) and endothelium (en).a, anterior; c, caudal; d, dorsal; r, rostral; p, posterior; v, ventral.Scale bars: 10 mm (a); 3 mm (b); 2 mm (c); 10 µm (d).

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I G U R E 2 Neoceratodus forsteri, ultrastructure of the surface and anterior regions of the corneal epithelium.(a) Scanning electron micrograph of the surface of the cornea showing a number of straight-sided cells containing a dense covering of microvilli and numerous holes.(b) Higher power micrograph of the microvilli and intervening holes.Note some regions are covered by a mucoid layer (asterisk).(c, d) Scanning electron micrographs of the edge of the central cornea showing palisades of vertical columns (c) within a single epithelial cell projecting to the surface.Mucous vesicles (asterisks) are shown in various regions along the columns.The thickness of the mucoid layer (ml) can also be seen.(e, f) Transmission electron micrographs showing the circular columns (c) in transverse (e) and tangential (f) planes.(g, h) Transverse sections of the epithelial surface showing the dense aggregations of the mucous vesicles (mv) in shallow (g) and deep (h) surface columns, which open at the surface to form a thick mucous layer (ml).Scale bars: 5 µm (a); 2 µm (b); 1 µm (c-e); 0.5 µm (f); 1 µm (g, h).

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7 of 24      the corneal surface (Figure2c-h).These cylindrical structures are 2.10 ± 0.92 µm tall and 405 ± 145 nm wide, when viewed with TEM (Figure2e-h).Within these cylindrical tubes and in the surrounding cytoplasm are numerous vesicles, which are 174.4 ± 153.3 nm in diameter and thought to contain mucus.Examination of these columnar structures using SEM (Figure2c,d) shows how they are arranged only along the cell surface.Measurements of the length of these structures (3.02 ± 0.48 µm) were significantly greater (p < .00001)than those measured using TEM, indicating that these measurements were not taken along the entire length of the columns.

F
I G U R E 3 Neoceratodus forsteri, ultrastructure of the corneal epithelium.(a) Low-power transmission electron micrograph of the stratified epithelial cell layers.(b) Higher power of a darkly staining superficial epithelial cell with a nucleus (n) surrounded by several endoplasmic reticula (er).(c) An intermediate epithelial cell showing the nucleus surrounded by tightly packed mucous vesicles (mv), which is attached to an adjacent cell via desmosomes (arrows).(d) Basement membrane (bm) underlying the epithelium (e) showing a number of hillocks (arrows) where collagen fibrils of the stroma protrude.B, Bowman's layer; ml, mucous layer; nc, nuclear convolution.Scale bars: 10 µm (a); 3 µm (b, c); 1 µm (d).
pigment epithelium(Figure 6d,e).The iris has an average thickness of 67.42 ± 14.66 µm, although it thins from around 98 µm in the periphery to 37 µm at the pupil margin.The majority of this difference occurs in the thickness of the irideal stroma, which is around 72 µm in thickness in the periphery, decreasing to 5 µm at the pupil margin, where it is replaced by a thickening of the pigment epithelia (Figures6d,e and 7a).The stroma of the iris is composed of loose connective tissue with bundles of collagen fibrils (with a diameter of 46.3 ± 9.2 nm), blood vessels and various cell configurations containing pigment granules (Figures6d,e and 7).In the anterior stroma and more markedly towards the periphery of the iris, there are several layers of thin cells packed with pigment granules (Figures6d and 7a,b).Along the anterior surface of the stroma are cells containing numerous straight-sided areas, where crystals (presumably containing guanine before fixation and histological processing) up to 4 µm in length lie in two to three layers, perpendicular to the incident light entering the eye (Figure7d,e,g).Within the iridial stroma, smooth muscle fibres

F
I G U R E 4 Neoceratodus forsteri, ultrastructure of the corneal stroma.(a) Low-power transmission electron micrograph of the stroma showing the alternating patterns of the collagen lamellae (cl) (dark where the collagen fibrils are running horizontally and light where the collagen fibrils are running perpendicularly).Note where groups of collagen fibrils branch and join other lamellae (arrows).(b-e) Series of high-power micrographs showing the variety of branching patterns ranging from only small numbers of fibrils (c, d) to large groups of fibrils (d, e).k, keratocyte.Scale bars: 3 µm (a); 1 µm (b); 0.5 µm (c-e).T A B L E 2 Summary of the various morphological features of the cornea across different classes of vertebrates.
microvilli, as opposed to the pattern of corneal microridges seen in a range of teleosts (H.B. Collin & S. P. Collin, 2000; S. P. Collin & H. B.
Abbreviations: AuL, autochthonous layer; B & A, branching and anastomosing of collagen lamellae in the stroma; DM, Desçemet's membrane; IL, iridescent layer; NR, not recorded in the original manuscript; NS, not stated in the original manuscript.a A divided cornea refers to the presence of two or more separated corneal stromas, including the presence of a primary, secondary or tertiary spectacle.b

F
I G U R E 6 Neoceratodus forsteri, ultrastructure of the corneal endothelium and iris.(a) Scanning electron micrograph of the surface of the endothelium showing interdigitation of apposing cell membranes (arrows).(b) Low-power transmission electron micrograph of the monocellular endothelium (en) underlying the stroma (s) in transverse section.(c) High-power micrograph of the junction of two endothelial cells (en) underlying a thin basement membrane (bm).(d) Light micrograph of the central region of the iris in transverse section showing anterior stroma (as) and posterior pigment epithelium (pe).Note the irideal stroma contains numerous rows of small pigment granules and collagen fibrils overlying large blood vessels (bv).(e) Low-power electron micrograph of the peripheral region of the posterior iris showing two layers of pigment granules within an anterior (ape) and a posterior (ppe) pigment epithelium.cl, collagen lamella; n, nucleus.Scale bars: 2 µm (a); 1 µm (b); 0.5 µm (c); 30 µm (d); 10 µm (e).
Neoceratodus forsteri, ultrastructure of the iris.(a) Light micrograph of the iris at the pupil margin in transverse section showing the anterior stroma (as) and pigment epithelium (pe).(b) Electron micrograph of the pigment epithelium divided into anterior (ape) and posterior (ppe) epithelial cell layers containing pigment granules.(c) Central iris showing bundles of collagen fibrils (cf) within the anterior stroma overlying the aggregations of pigment granules within the anterior pigment epithelium (ape).(d) High power of an anterior cell containing rectangular crystals that may have contained guanine (gc) before histological processing.(e) Lower power of two cells putatively containing guanine crystals (gc) that have separated from the anterior surface of the iris.(f) Tangential section of the iris at the level of the pigment epithelium showing narrow lateral extensions of many of the pigment granules (arrows).(g) Anterior cell containing stacks of guanine crystals (gc) overlying pigment granules (pg).n, nucleus.Scale bars: 50 µm (a); 2 µm (b); 3 µm (c); 0.5 µm (d); 2 µm (e, f); 1 µm (g).

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I G U R E 8 Neoceratodus forsteri, ultrastructure of the peripheral iris.(a) Low-power electron micrograph showing a number of peripheral cells (pc) within the anterior stroma.(b) High power of a peripheral cell containing many lobular structures (ls).(c) Convolutions of the basement membrane (bm) in a region anterior to the anterior pigment epithelium.(d).High power of lobular structures (ls) and a membranous inclusion (asterisk) within the cytoplasm of a peripheral cell.ape, anterior pigment epithelium; n, nucleus; ppe, posterior pigment epithelium.Scale bars: 10 µm (a); 2 µm (b, c); 0.3 µm (d).
densities of large epithelial cells to assist with the osmotic stress placed on the cornea when exposed to different environmental conditions and the homeostatic control of water content to maintain transparency.The epithelial cells release mucous granules onto the corneal surface to avoid desiccation while on land and are covered in microvilli to increase the cell surface area, which is vital for assisting the transport of nutrients and oxygen to the cornea.A Bowman's layer is present and with branching of collagen lamellae within the stroma, would help to minimise swelling of the stroma during its migrations onto land.The absence of a Desçemet's membrane and an annular ligament to support the curvature of the cornea and iris, respectively, may not be as important for N. forsteri given its low reliance on vision.The structure of the cornea reflects a range of adaptations for amphibious behaviour and is more similar to nonaquatic tetrapods than to bony fishes.The iris is well developed and contractile, a trait not common in fishes, with dense aggregations of pigment and occasional deposits of reflective material giving rise to its colour.AUTHOR CONTRIBUTIONSHermann Barry Collin and Shaun P. Collin designed the study, were responsible for most of the tissue preparation and ultrastructural analyses and interpretation of the results.Julian Ratcliffe performed parts of the histological analyses and all authors assisted in the writing of the manuscript.