This study provides a large and phylogenetically very broad examination of the thickness of the snake spectacle in various species with different habitats and activity patterns. Spectacle thickness was found to vary significantly between species. The average thickness ranged between 74 and 244 μm. The thinnest spectacle belonged to a viper (Viperidae), the thickest to a pipe snake (Cylindrophiidae). Species variation of the spectacle has also recently been reported by Van Doorn and Sivak [35] that found that vipers generally have thinner spectacle scales than colubrids. However, that study examined only the keratin layers of the spectacle which are periodically shed along with the skin of the body, whereas this present study was able to include the spectacle itself, the structure which remains permanently fixed to the snake.
The examined snakes were all alcohol-preserved museum specimens. Museum specimens have been validated in other studies [5], and they provided an opportunity to examine a wide range of different species. Some of the specimens had been preserved in ethanol for more than 100 years, however, the quality of the scanning images was remarkable. A previous study displayed no significant difference in spectacle thickness between live and formalin fixed specimens [7]. The specimens examined in this study were fixed in ethanol and studies have shown that morphology of ethanol fixed tissues are comparable to formalin fixed tissues so tissue shrinkage was not considered a concern [36, 37]. In either case all specimens included were handled similarly, so that fixation would not have introduced bias to the study. Some of the examined species had extremely small eyes making it difficult to obtain a representable image as the equipment is based on examinations of the human eye. However, images of even the smallest eye were able to be enlarged on the computer screen using the built-in software so measurements of spectacle thickness were possible. Measurements were made centrally on the spectacle proper. A previous study showed that spectacle thickness is uniform throughout the spectacle proper, but increases in the peripheral region known as the transition zone [7].
The multivariate analysis showed that the variation of spectacle thickness was predicted by taxonomic family and habitat in contrast to spectacle diameter, which was correlated to all three parameters: taxonomic family, habitat and activity pattern. This indicates for example, that diurnal snakes have large eyes but variable spectacle thickness.
When looking at spectacle thickness from a habitat point-of-view, it was found that arboreal and terrestrial snakes had thin spectacles and fossorial and aquatic snakes thick spectacles. This supports the idea that the spectacle has a protective function [3, 35] and that snakes surrounded by water, or living underground need a stronger protective layer than arboreal and terrestrial snakes. From a vision point of view, it could mean that arboreal and terrestrial snakes have a different need for vision than the aquatic and fossorial snakes. Fossorial or burrowing snakes live mainly in the dark and their need for vision is very limited. The aquatic snakes have lost the refractive power of the anterior surface of the eye as a result of a high refractive index of water [38]. On the other hand, terrestrial and arboreal snakes may have developed a thinner spectacle to improve visual acuity, regardless of whether they stem from fossorial [39] or aquatic [40, 41] ancestors. Besides varying needs for vision, another possibility could be the development of a different mechanism of accommodation. Snakes typically focus by moving their rigid lens towards or away from the retina [42] in comparison to mammals where the lens is deformable [43]. It has been stated that the lens of the aquatic dice snake (Natrix tesselata) is very flexible compared to other snakes which would be perfectly suited for large degrees of accommodation [44, 38]. The exact mechanisms have, however, not been studied.
Diurnal snakes had significantly larger eyes than nocturnal snakes, a feature contrary to the eyes of mammals, where nocturnal individuals possess the larger eyes. These findings are in accordance with a recent study on colubrid snakes [5]. Snakes with large horizontal spectacle diameter have a larger radius of curvature than snakes with small eyes [5]; and assuming all other factors are equal, the refractive ability (\( F=\frac{n^1-n}{r} \), where r = radius of curvature, n = refractive index of medium which light is passing from, n1 = refractive index of medium into which light is passing) of a large spectacle is lower than that of a small spectacle [45]. Thus, in diurnal snakes, the lens needs to assume a more powerful refractive role than in nocturnal snakes in order to achieve the same overall refractive power of the eye, as mentioned above in the aquatic dice snake. Furthermore, the cornea could also play a role that is yet to be discovered. Additional investigations of snake vision are required to further elaborate on the refractive power of the snake eye.
The spectacle is found in all snakes, which suggests that it is an adaptive trait, which has been conserved through snake diversification, but its adaptive significance is still unknown. This study shows a large interspecific variation in spectacle morphology among snakes, which may indicate that the spectacle does not serve the same function in all species. We show that ancestral snakes had thick spectacles and were fossorial supporting the idea that the incipient function of the spectacle was eye protection. Since spectacle thickness was secondarily reduced multiple times independently may suggest that an evolutionary trade-off exists between eye protection and other functions, such as vision, resulting in the observed variation in spectacle morphology between extant snake species.
