2 Materials and methods
The materials used in the present study were obtained from snake specimens stored in ethanol 70% in the reptile collection of the Razi University Zoological Museum (RUZM) and the Zoological Collection of Reza Babaie Savasari (ZMRBS). 22 adult snake specimens from seven species were investigated. The selected snakes included six viperid and two colubrid species. These snakes are found in different regions and inhabit more or less different habitats in the Middle East. The Viperinae or old world vipers are a subfamily of venomous snakes endemic to Europe, Asia, and Africa. The Viperinae genus PseudocerastesBoulenger, 1896, comprises three species; Pseudocerastes persicus (Duméril, Bibron & Duméril, 1854), Pseudocerastes fieldi Schmidt, 1930 andPseudocerastes urarachnoides Bostanchi, Anderson, Kami & Papenfuss, 2006. The specimens used in this study were collected from Kerman Province, Iran for P. persicus , from southwestern Iraq forP. fieldi , and from Ilam province in western Iran for P. urarachnoides . The monotypic genus Eristicophis , represented byE. macmahonii Alcock & Finn1897, was captured in Nushki province, southwestern Pakistan. Genus Cerastes , represented byCerastes gasperettii Leviton & Anderson, 1967, was captured in Khuzestan province, southwestern Iran, and al-Diwaniyah Governorate, Iraq. Genus Echis represented by E. carinatus which was collected in the mountainous areas of the northwest of Kerman province. Family Colubridae Oppel, 1811, which is the largest snake family consists of aglyphous and opisthoglyphous snakes, represented by two genera EirenisJan, 1863 andLytorhynchusPeters, 1862 and two species in this study: Eirenis rafsanjanicus Akbarpour, Fathinia, Rastegar-Pouyani & Rastegar-Pouyani, 2020 collected from Kerman province in the south-central Iran, and Lytorhynchus maynardi Alcock & Finn, 1897 from Zabol county, Sistan & Balouchistan province, southeastern Iran.
To investigate scale’s microornamentation, dorsal scale samples of each specimen cut off from the head, mid-part of the body, and tail. The scales were placed in encoded tubes and filled with distilled water and neutral soap. The tubes were manually shaken for about one minute to remove probable impurities, followed by washing with distilled water and left to dry at room temperature for about 5 minutes. At next step, the scales were mounted on SEM rotatable aluminum stubs with a thin veneer of glue. Stubs were coated with gold and photographed using a scanning electron microscope (Hitachi S-2460 N SEM) at 25kV under various magnifications (range: 20x–10000x).
To compare the light reflected by the surface of scales, snakes were examined adopting two different methods: firstly, the whole body of the snake was photographed at different wavelengths of light using FluoVision Imaging System, and secondly, thick pieces of the snake’s dorsal skin were prepared in the same way mentioned above for microornamentation study. The skin pieces were mounted on the girdles with glue. Skin reflectance was measured in the 300‒700 nm (UV-VIS) spectral ranges using a Lambda2S spectrometer (Perkin Elmer, USA).