Patterns of morphological variation and ecological correlates in the skull of vipers (Serpentes: Viperidae).

The skull of vipers is a highly kinetic anatomical structure involved in envenomating and consuming of prey. Morphological knowledge about the viperid skull is based on studies on some groups of species, but information on its variation within the whole family and its functional morphology is still scarce. In this study, we aimed to explore variation in skull morphology among species of the three subfamilies of Viperidae, and test whether that variation correlates with macrohabitat and diet. We performed quantitative analyses of the viperid skull based on broad taxonomic sampling and two methodological approaches: linear and geometric morphometrics. The results of both approaches showed that much of the variation lies in differences of shape and relative size of the premaxilla, the nasals, the frontals, and the parietals. The results indicated that phylogeny and size influence the shape of the skull, but we also found evidence of morphological differentiation between arboreal and terrestrial species and in species with mammal specialist diet. Our findings imply that, besides evolutionary allometry and phylogenetic signal, demands of particular diets coupled with use of certain habitats have in part shaped morphological evolution of the viperid skull.

Evolutionary allometry and ecological correlates of fang length evolution in vipers.

Traits for prey acquisition form the phenotypic interface of predator-prey interactions. In venomous predators, morphological variation in venom delivery apparatus like fangs and stingers may be optimized for dispatching prey. Here, we determine how a single dimension of venom injection systems evolves in response to variation in the size, climatic conditions and dietary ecology of viperid snakes. We measured fang length in more than 1900 museum specimens representing 199 viper species (55% of recognized species). We find both phylogenetic signal and within-clade variation in relative fang length across vipers suggesting both general taxonomic trends and potential adaptive divergence in fang length. We recover positive evolutionary allometry and little static allometry in fang length. Proportionally longer fangs have evolved in larger species, which may facilitate venom injection in more voluminous prey. Finally, we leverage climatic and diet data to assess the global correlates of fang length. We find that models of fang length evolution are improved through the inclusion of both temperature and diet, particularly the extent to which diets are mammal-heavy diets. These findings demonstrate how adaptive variation can emerge among components of complex prey capture systems.

Identification of the myodural bridge in a venomous snake, the gloydius shedaoensis: what is the functional significance? / Identificación del puente miodural en una serpiente venenosa, gloydius shedaoensis: ¿cuál es el significado funcional?

SUMMARY: Myodural bridges (MDB) are anatomical connections between the suboccipital muscles and the cervical dura mater which pass through both the atlanto-occipital and the atlanto-axial interspaces in mammals. In our previous studies, we found that the MDB exists in seven terrestrial mammal species, two marine mammal species, two reptilian species, and one bird species. A recent study suggested that given the "ubiquity" of myodural bridges in terrestrial vertebrates, the MDB may also exist in snakes. Specifically, we focused on the Gloydius shedaoensis, a species of Agkistrodon (pit viper snake) that is only found on Shedao Island, which is in the southeastern sea of Dalian City in China. Six head and neck cadaveric specimens of Gloydius shedaoensis were examined. Three specimens were used for anatomical dissection and the remaining three cadaveric specimens were utilized for histological analysis. The present study confirmed the existence of the MDB in the Gloydius shedaoensis. The snake's spinalis muscles originated from the posterior edge of the supraoccipital bones and the dorsal facet of the exocciput, and then extended on both sides of the spinous processes of the spine, merging with the semispinalis muscles. On the ventral aspect of this muscular complex, it gave off fibers of the MDB. These MDB fibers twisted around the posterior margin of the exocciput and then passed through the atlanto-occipital interspace, finally terminating on the dura mater. We observed that the MDB also existed in all of the snakes' intervertebral joints. These same histological findings were also observed in the Gloydius brevicaudus, which was used as a control specimen for the Gloydius shedaoensis. In snakes the spinal canal is longer than that observed in most other animals. Considering the unique locomotive style of snakes, our findings contribute to support the hypothesis that the MDB could modulate cerebrospinal fluid (CSF) pulsations.

RESUMEN: Los puentes miodurales (MDB) son conexiones anatómicas entre los músculos suboccipitales y la duramadre cervical que pasan a través de los espacios intermedios atlanto-occipital y atlanto-axial en los mamíferos. En nuestros estudios anteriores, encontramos que el MDB existe en siete especies de mamíferos terrestres, dos especies de mamíferos marinos, dos especies de reptiles y una especie de ave. Un estudio reciente sugirió que dada la "ubicuidad" de los puentes miodurales en los vertebrados terrestres, el MDB también puede existir en las serpientes. Específicamente, nos enfocamos en Gloydius shedaoensis, una especie de Agkistrodon (serpiente víbora) que solo se encuentra en la isla Shedao, en el mar sureste de la ciudad de Dalian en China. Se examinaron seis especímenes cadavéricos de cabeza y cuello de Gloydius shedaoensis. Se utilizaron tres especímenes para la disección anatómica y los tres especímenes cadavéricos restantes se utilizaron para el análisis histológico. El presente estudio confirmó la existencia del MDB en Gloydius shedaoensis. Los músculos espinosos de la serpiente se originaron en el margen posterior de los huesos supraoccipital y la cara dorsal del exoccipucio, y luego se extendieron a ambos lados de los procesos espinosas de la columna vertebral, fusionándose con los músculos semiespinosos. En la cara ventral de este complejo muscular se desprendían fibras del MDB. Estas fibras MDB se ubican alrededor del margen posterior del exoccipucio y luego atraviesan el interespacio atlanto-occipital, terminando finalmente en la duramadre. Observamos que el MDB también existía en todas las articulaciones intervertebrales de las serpientes. Estos mismos hallazgos histológicos también se observaron en Gloydius brevicaudus, que se utilizó como muestra de control para Gloydius shedaoensis. En las serpientes, el canal espinal es más largo que el observado en la mayoría de los otros animales. Teniendo en cuenta el estilo único locomotor de las serpientes, nuestros hallazgos contribuyen a respaldar la hipótesis de que el MDB podría modular las pulsaciones del líquido cerebroespinal.

Venom Gland Mass Spectrometry Imaging of Saw-Scaled Viper, Echis carinatus sochureki, at High Lateral Resolution.

The snake venom gland is the place for the synthesis, storage, and secretion of a complex mixture of proteins and peptides, i.e., the venom. The morphology of the gland has been revealed by classical histology and microscopic studies. However, knowledge about the gland's cellular secretory and functional processes is still incomplete and has so far been neglected by the omics disciplines. We used autofocusing atmospheric-pressure matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) to investigate endogenous biomolecular distributions in the venom glands of the saw-scaled viper, Echis carinatus sochureki, employing different sample preparation methods. Fresh-freezing and formalin-fixation were tested for the gland to obtain intact tissue sections. Subsequently, MSI was conducted with 12 µm pixel resolution for both types of preparations, and the lateral distributions of the metabolites were identified. Experiments revealed that lipids belonging to the classes of PC, SM, PE, PS, PA, and TG are present in the venom gland. PC (32:0) and SM (36:1) were found to be specifically located in the areas where cells are present. The snake venom metalloprotease inhibitor pEKW (m/z 444.2233) was identified in the venom by top-down LC-MS/MS and localized by MALDI-MSI in the gland across secretory epithelial cells. The peptide can inhibit the venom's enzymatic activity during long-term storage within the venom gland. With a high degree of spectral similarities, we concluded that formalin-fixed tissue, in addition to its high ability to preserve tissue morphology, can be considered as an alternative method to fresh-frozen tissue in the case of lipid and peptide MS imaging in venom gland tissues.

Skull osteology of Vipera walser (Squamata, Viperidae): Description, variability, ontogeny, and diagnostic characters in comparison to other Italian vipers.

Vipera walser is the most recently recognized European viper. This rare species is endemic to a small area in the Piedmont Alps of Italy, but its closest relatives are found among the Caucasian viper species. In order to provide a starting point for a phylogenetic and biogeographic investigation based on osteology, and including fossils remains, we analyzed four specimens of V. walser and compared them with specimens of the four other Italian viper species. Based on these specimens, we improved the diagnosis of V. walser and provided a first evaluation of intraspecific variability and ontogenetic variation. The skull of V. walser is subject to significant variation, most likely related to ontogeny in some cases (i.e., development of the parietal crest, development of the basioccipital process, shape of the posterior margin of the parabasisphenoid, shape of the quadrate). Based on the studied material, it is possible to distinguish V. walser from the other Italian vipers by the shape of the occipital crest of the supraoccipital, which is posteriorly directed, whereas it is laterally directed in the other species. The osteological diagnosibility provides further support for the validity of V. walser as a distinct species from Vipera berus.

Arboreality constrains morphological evolution but not species diversification in vipers.

An increase in ecological opportunities, either through changes in the environment or acquisition of new traits, is frequently associated with an increase in species and morphological diversification. However, it is possible that certain ecological settings might prevent lineages from diversifying. Arboreality evolved multiple times in vipers, making them ideal organisms for exploring how potentially new ecological opportunities affect their morphology and speciation regimes. Arboreal snakes are frequently suggested to have a very specialized morphology, and being too large, too small, too heavy, or having short tails might be challenging for them. Using trait-evolution models, we show that arboreal vipers are evolving towards intermediate body sizes, with longer tails and more slender bodies than terrestrial vipers. Arboreality strongly constrains body size and circumference evolution in vipers, while terrestrial lineages are evolving towards a broader range of morphological variants. Trait-dependent diversification models, however, suggest similar speciation rates between microhabitats. Thus, we show that arboreality might constrain morphological evolution but not necessarily affect the rates at which lineages generate new species.

Morphology of the snake spectacle reflects its evolutionary adaptation and development.

BACKGROUND: Covering the eye of all snakes is a transparent integumental structure known as the spectacle. In order to determine variations in spectacle thickness among species, the spectacles of 217 alcohol-preserved museum specimens of 44 species belonging to 14 different families underwent optical coherence tomography (OCT) to measure spectacular thickness. Multivariable analyses were made to determine whether family, activity period (diurnal/nocturnal) and habitat (arboreal/terrestrial/fossorial/aquatic) influenced spectacle thickness. RESULTS: The thinnest spectacles in absolute terms were found in the Usambara bush viper (Viperidae) with a thickness of 74 ± 9 µm and the absolute thickest spectacle was found in the red-tailed pipe snake (Cylindrophiidae) which had a spectacle thickness of 244 ± 57 µm. Fossorial and aquatic snakes had significantly thicker spectacles than arboreal and terrestrial snakes. When spectacle thickness was correlated to eye size (horizontal spectacle diameter), Gray's earth snake (Uropeltidae) had the lowest ratio (1:7) and the cottonmouth (Viperidae) had the highest ratio (1:65). Multivariable and phylogenetic analyses showed that spectacular thickness could be predicted by taxonomic family and habitat, but not activity period. CONCLUSION: This phylogenetically broad systematic study of the thickness of the snake spectacle showed that spectacular thickness varies greatly across snake species and may reflect evolutionary adaptation and development.

Venom yield and its relationship with body size and fang separation of pit vipers from Argentina.

The amount of venom that a snake can inject is related to its body size. The body size is related to head size and to the distance between fangs. To correlate snake body size, distance between fangs and distance between puncture wounds with the venom yield (and consequently with the venom dose potentially injected in a single snakebite), we studied these variables in two species of public health importance in South America, Bothrops (Rhinocerophis) alternatus, and Crotalus durissus terrificus. In all cases a positive correlation was observed between body length, fang separation distance, distance between puncture wounds and venom yield, with a regression coefficient over 0.5 for Bothrops alternatus and over 0.6 for Crotalus durissus terrificus in all cases, being the relation distance between punctures wounds and venom yield of 0.54 and 0.69 respectively. The difference between fang separation and puncture separation was never greater than 30%, with a mean difference around 13%. The strong relationships between body size, fang separation and venom yield may be useful for planning potential venom production in serpentariums. In addition, because puncture mark separation gives an approximate idea of the size of the snake, this provides a rough idea of the size of the snake that produced a bite and the potential amount of venom that could have been injected.

A new large species of Bitis Gray, 1842 (Serpentes: Viperidae) from the Bale Mountains of Ethiopia.

A new species of viperine viperid snake is described, Bitis harenna sp. nov. The new species is a member of the subgenus Macrocerastes based on it having three scales separating the nasal and rostral shields, and on the combination of 'divisions' of dorsal scale rows on the upper flanks and 'fusions' of rows on the lower flanks. Bitis harenna sp. nov. is distinguished from other members of the subgenus by its unique colour pattern, posterior parietal flange on the lateral wall of the braincase, and possibly by differences in scalation and head proportions. Only a single museum specimen is known, a female collected from 'Dodola' in Ethiopia probably in the late 1960s and previously identified as a possibly unusually coloured and patterned B. parviocula. A live, presumably male, specimen very closely resembling the holotype of Bitis harenna sp. nov. was photographed on the Harenna escarpment of the Bale Mountains National Park, Ethiopia in 2013, providing secure occurrence data and evidence that the holotype is not a uniquely aberrant specimen. A revised key to the species of Bitis in Ethiopia is presented. Aspects of body scalation are compared among species of the subgenus Macrocerastes and between species of Macrocerastes and Bitis, and several systematic characters are highlighted and clarified.

Multilocus phylogeny of the Asian Lance-headed pitvipers (Squamata, Viperidae, Protobothrops).

Despite the public health significance and potential applications to medical research, the evolutionary history of the Asian Lance-headed pitvipers (genus Protobothrops) remains inadequately studied. Previous research generally focused on a few selected species with no comprehensive molecular study of Protobothrops. Here, we conduct the first study to infer the phylogenetic relationships of all currently recognized species in this genus based on four mitochondrial DNA fragments and four nuclear genes. Bayesian Inference and Maximum Likelihood analyses show that within Protobothrops there are four strongly supported clades forming distinct subgroups. The first subgroup, which is sister to the other three, consists of three species, P. himalayanus, P. kaulbacki, and P. sieversorum. The second subgroup contains only P. mangshanensis. The final two subgroups, which are sister groups, include the other four and six Protobothrops species. Although our findings provide additional information on the phylogenetic relationships of the genus Protobothrops, the placement of P. dabieshanensis and P. elegans remains problematic. In addition, our molecular results indicate that P. trungkhanhensis, currently considered endemic to Vietnam, should be added to the species known from China. Our ancestral area estimation indicated that Protobothrops likely originated in southwestern China. This study improves our understanding of the evolutionary relationships among species of Asian Lance-headed pitvipers, providing a greater framework for future studies.

A cryptic palm-pitviper species (Squamata: Viperidae: Bothriechis) from the Costa Rican highlands, with notes on the variation within B. nigroviridis.

Middle America is one of the most biodiverse regions in the world, harboring an exceptional number of rare and endemic species. This is especially true of Middle American cloud forests, where montane specialists occupy restricted, high-elevation ranges making them attractive candidates for investigating historical biogeography and speciation. One such highland-restricted species, the black speckled palm-pitviper (Bothriechis nigroviridis), occupies the Central, Tilarán, and Talamanca Cordilleras in Costa Rica and Panama. In this study, we investigate the genetic and morphological variation among populations of B. nigroviridis by inferring a multilocus phylogeny (21 individuals) and analyzing meristic scale characters with a principal component analysis (64 individuals). We find B. nigroviridis sensu stricto to be composed of two deeply divergent lineages, one with a restricted range in the northern and central Cordillera Talamanca and the other ranging throughout the Central, Tilarán, and Talamanca Cordilleras. Furthermore, these two lineages are morphologically distinct, with previously unrecognized differences in several characters allowing us to name and diagnose a new species B. nubestris sp. nov. We also examine the genetic and morphological variation within B. nigroviridis and discuss biogeographic hypotheses that may have led to the diversification of Bothriechis lineages.

Eye and pit size are inversely correlated in crotalinae: Implications for selection pressure relaxation.

Mate, prey, and predator recognition often depend on the integration of information from multiple sensory modalities including visual, auditory, and/or olfactory inputs. In Crotalinae, the eyes sense visible light while the pit organs detect infrared (IR) radiation. Previous studies indicate that there is significant overlap between the eye and pit sensory fields and that both senses are involved in recognition processes. This study investigated the relationships between eye and pit sizes in this taxonomic group as a function of phylogeny and habitat. In view of the fact that pit orientation depends largely on snout shape, pit vipers were grouped as follows: 1) arboreal, 2) terrestrial with rounded snout, and 3) terrestrial with pointed snout. The pit orientations and habitant patterns were fully independent of the Crotalinae phylogenetic tree. The phylogenetic generalized least squares model showed that both eye and pit areas were not of significantly phylogenetic relatedness, implying alternatively a strong effect of adaptation on eye and pit sizes. Negative correlations between relative eye and pit areas in terrestrial (both pointed and rounded snouts) and arboreal species were statistically significant. Our results suggest that the eyes and pits function in a complementary fashion such that selection for IR-perception relaxes selection pressures on the visual system and selection for visual discrimination relaxes selection pressures acting on the IR-system.

Discovery of a novel accessory structure of the pitviper infrared receptor organ (serpentes: viperidae).

The facial pits of rattlesnakes, copperheads, lanceheads, bushmasters and other American and Asian pitvipers (Crotalinae) are highly innervated and densely vascularized infrared (IR) receptor organs. For over a century, studies have focused on a small sample of model species from North America and Asia. Based on an expanded survey of Central and South American crotalines, we report a conspicuous accessory structure composed of well-defined papillae that project from the anterior orbital adnexa. The papillae are continuous with the inner chamber of the IR receptor organ and our histological and ultrastructural data suggest that they possess a well-developed nervous network and extensive vascularization; however, they lack the characteristic IR-sensitive terminal nerve masses found in the IR-receptive pit membrane. The function of the IR receptor organ papillae is unknown.

Non-contaminating camouflage: multifunctional skin microornamentation in the West African Gaboon viper (Bitis rhinoceros).

The West African Gaboon viper (Bitis rhinoceros) has an extraordinary coloration of pale brown and velvety black markings. The velvety black appearance is caused by a unique hierarchical surface structures which was not found on the pale brown scales. In the present study we examined the wettability of the vipers scales by measuring contact angles of water droplets. Velvet black scale surfaces had high static contact angles beyond 160° and low roll-off angles below 20° indicating an outstanding superhydrophobicity. Our calculations showed that the Cassie-Baxter model describes well wettability effects for these surfaces. Self-cleaning capabilities were determined by contaminating the scales with particles and fogging them until droplets formed. Black scales were clean after fogging, while pale scales stayed contaminated. Black scales feature multifunctional structures providing not only water-repellent but also self-cleaning properties. The pattern of nanoridges can be used as a model for surface-active technical surfaces.

Snake velvet black: hierarchical micro- and nanostructure enhances dark colouration in Bitis rhinoceros.

The West African Gaboon viper (Bitis rhinoceros) is a master of camouflage due to its colouration pattern. Its skin is geometrically patterned and features black spots that purport an exceptional spatial depth due to their velvety surface texture. Our study shades light on micromorphology, optical characteristics and principles behind such a velvet black appearance. We revealed a unique hierarchical pattern of leaf-like microstructures striated with nanoridges on the snake scales that coincides with the distribution of black colouration. Velvet black sites demonstrate four times lower reflectance and higher absorbance than other scales in the UV-near IR spectral range. The combination of surface structures impeding reflectance and absorbing dark pigments, deposited in the skin material, provides reflecting less than 11% of the light reflected by a polytetrafluoroethylene diffuse reflectance standard in any direction. A view-angle independent black structural colour in snakes is reported here for the first time.

Shape Variation and Allometry in the Precloacal Vertebral Series of the Snake Daboia russelli (Viperidae) / Variación Morfológica y Alometría de las Vértebras Precloacales en el Ofidio Daboia russelli (Viperidae)

Understanding the variation of the ophidian vertebral morphology is an essential tool in snake paleobiology, but so far this field remains hardly investigated. A major problematic is the still scarce knowledge about the basis of homogeneity of intracolumnar shape variation along the vertebrae of the precloacal region in these animals. For instance, this variation can be overwhelmingly low in cases such as in vipers, for which it seems almost impossible to describe a concrete regionalization of the precloacal region without ambiguity. This study has applied geometric morphometrics to analyze if the shape variation of the vertebrae of the precloacal vertebrae of an adult specimen of Daboia russelli allows differentiating any sort of parcellation within the column of this organism. We have also explored if size is associated with the organization of vertebral shape along the axial skeleton. The multivariate analyses showed that the main pattern of vertebral shape variation in D. russelli concerns the neural spine and the hypapophysis, whereas the shape of the vertebral centrum appears to be nearly invariant along the series. Our analysis also showed that the precloacal region can be sudivided into two portions that merge in a transitional boundary of largest vertebrae in the middle of the column. From this middle region towards the distal ends of the column vertebrae become smaller changing their shapes in two antithetical ways.

Entender la variación en la morfología vertebral de los ofidios es crucial para la paleobiología del grupo pero, hasta ahora, este campo está poco investigado. Uno de los principales problemas es el escaso conocimiento sobre las bases de la homogeneidad en la variación de la forma a lo largo de la región precloacal en estos animales. Por ejemplo, en el caso de las víboras, dicha variación puede ser muy pequeña lo cual hace casi imposible la descripción de una regionalización precisa sin ambigüedad. En este estudio se ha aplicado morfometría geométrica para analizar si la variación de la forma vertebral de un individuo adulto de la especie Daboia russelli permite subdividir la región precloacal. Además, hemos explorado si el tamaño está asociado con la organización vertebral a lo largo del esqueleto axial. Los análisis multivariantes han demostrado que el patrón principal de la variación de la forma vertebral está determinado por la espina neural y la hipapofisis, mientras que el centro vertebral varía poco a lo largo de la serie. Nuestro análisis ha mostrado que la región precloacal puede ser dividida en dos series cuya separación está marcada por las vértebras más grandes, posicionadas aproximadamente en la mitad de la columna. Tomando como referencia la mitad de la columna, hacia los extremos distales, las vértebras tienden a ser más pequeñas cambiando su forma de modo antitético.

Analytical methods for the geometric optics of thermal vision illustrated with four species of pitvipers.

The pitviper facial pit is a pinhole camera-like sensory organ consisting of a flask-shaped cavity divided into two chambers by a suspended membrane. Neurophysiological studies and simplified optical models suggest that facial pits detect thermal radiation and form an image that is combined with visual input in the optic tectum to form a single multispectral image. External pit anatomy varies markedly among taxonomic groups. However, optical function depends on unknown internal anatomy. Therefore, we developed methods for relating anatomy to optical performance. To illustrate, we constructed detailed anatomical models of the internal anatomy of the facial pits of four individuals of four pitviper species using X-ray tomography sections of fresh material. We used these models to define the point spread function, i.e. the distribution of radiation from a point source over the pit membrane, for each species. We then used optical physics, heat transfer physics and computational image processing to define the thermal image formed on the pit membrane for each species. Our computed pit membrane images are consistent with behavioral observations if the sensitivity of membrane receptors equals the most sensitive (ca. 0.001°C) laboratory estimates. Vignetting (variation in optical aperture size with view angle) and differences between body and environmental temperatures can create temperature variation across the membrane that greatly exceeds image temperature contrasts, potentially impairing imaging. Spread functions plotted versus source point azimuth and elevation show distinct patterns that suggest new research directions into the relationships among the optical anatomy, ecology, behavior and sensory neurophysiology of pitvipers.

Reduced performance of prey targeting in pit vipers with contralaterally occluded infrared and visual senses.

Both visual and infrared (IR) senses are utilized in prey targeting by pit vipers. Visual and IR inputs project to the contralateral optic tectum where they activate both multimodal and bimodal neurons. A series of ocular and pit organ occlusion experiments using the short-tailed pit viper (Gloydius brevicaudus) were conducted to investigate the role of visual and IR information during prey targeting. Compared with unoccluded controls, snakes with either both eyes or pit organs occluded performed more poorly in hunting prey although such subjects still captured prey on 75% of trials. Subjects with one eye and one pit occluded on the same side of the face performed as well as those with bilateral occlusion although these subjects showed a significant targeting angle bias toward the unoccluded side. Performance was significantly poorer when only a single eye or pit was available. Interestingly, when one eye and one pit organ were occluded on opposite sides of the face, performance was poorest, the snakes striking prey on no more than half the trials. These results indicate that, visual and infrared information are both effective in prey targeting in this species, although interference between the two modalities occurs if visual and IR information is restricted to opposite sides of the brain.

Comparative morphology of the skin of Natrix tessellata (family: Colubridae) and Cerastes vipera (family: Viperidae).

We studied beneficial difference of the skin of two snakes. Two snakes were chosen from two different habitats and two families: Colubridae (Natrix tessellata) and Viperidae (Cerastes vipera). The investigations were performed by light and electron microscopy. Histologically, the skin of the studied species show pronounced modifications that correlated with functional demands. The scales in Natrix tessellata overlapped slightly, while in Cerastes vipera they were highly overlapped. SEM shows that scales of Natrix tessellata had bidentate tips while the scales of Cerastes vipera were keeled. Histochemically, in both studied species, melanocytes and collagenous fibres were distributed throughout the dermis. Polysaccharides were highly concentrated in the epidermis and dermis of both species while proteins were highly concentrated only in the epidermis. Transmission electron microscopy (TEM) showed that the skin of both snakes consisted of keratins located in the epidermis. Some lipids and mucus were incorporated into the outer scale surfaces such that lipids were part of the fully keratinised hard layer of the snakes' skins. Lipids are probably responsible for limiting water loss and ion movements across the skin. Melanosomes from epidermal melanocytes were present only in Cerastes vipera. In aggregate, these results indicate that snakeskin may provide an ecological indicator whereby epidermal and integumentary specializations may be ecologically correlated.