Life Science Journal, 2012;9(1) http://www.lifesciencesite.com Genetic Diversity among Eight Egyptian Snakes (Squamata-Serpents: Colubridae) Using RAPD-PCR Nadia H. M. Sayed Zoology Dept., College for Women for Science, Arts and Education, Ain Shams University, Heliopolis, Cairo, Egypt. [email protected] Abstract: Genetic variations between 8 Egyptian snake species, Psammophis sibilans sibilans, Psammophis Sudanensis, Psammophis Schokari Schokari, Psammophis Schokari aegyptiacus, Spalerosophis diadema, Lytorhynchus diadema, , Coluber rhodorhachis, Coluber nummifer were conducted using RAPD-PCR. Animals were captured from several locality of Egypt (Abu Rawash-Giza, Sinai and Faiyum). Obtained results revealed a total of 59 bands which were amplified by the five primers OPB-01, OPB-13, OPB-14, OPB-20 and OPE-05 with an average 11.8 bands per primer at molecular weights ranged from 3000-250 bp. The polymorphic loci between both species were 54 with percentage 91.5 %. The mean band frequency was 47% ranging from 39% to 62% per primer .The similarity matrix value between the 8 Snakes species was ranged from 0.35 (35%) to 0.71 (71%) with an average of 60%. The genetic distance between the 8 colubrid species was ranged from 0.29 (29%) to 0.65 (65%) with an average of 40 %. Dendrogram showed that, the 8 snake species are separated from each other into two clusters .The first cluster contain 4 species of the genus Psammophis. The second cluster includes the 4 species of the genera, Spalerosophis; Coluber and Lytorhynchus. Psammophis sibilans is sister to Psammophis Sudanensis with high genetic similarity (71%) and Psammophis Schokari Schokari is sister to Psammophis Schokari aegyptiacus with high genetic similarity (70%). The Coluber rhodorhachis are clustered and closer to Spalerosophis diadema (70%) than to Coluber nummifer (57%). Therefore, the evolutionary history of snakes still remains controversial. It is concluded that, the similarity coefficient and the genetic distance value between the 8 snake species indicates that, the 8 snake species are not identical and separated from each other. [Nadia H. M. Sayed Genetic Diversity among Eight Egyptian Snakes (Squamata-Serpents: Colubridae) Using RAPD-PCR. Life Science Journal 2012; 9(1):423-430]. (ISSN: 1097-8135). http://www.lifesciencesite.com. 63 Key Words: Colubridae, Serpents, RAPD-PCR, Phylogenetic Relationship, Egyptian snakes. 1. Introduction studies (Pinou and Dowling, 1994), biochemical The squamates are the most diversified group electrophoresis (Cadle, 1988; Dowling et al., 1996) containing the lizards and snakes (Vidal and Hedges, and molecular sequence analysis (Lawson et al., 2005; 2009). Several investigations have been recorded on Burbrink and Pyron, 2008; Wiens et al., 2008; Kelly the fauna of Egypt reptiles (Anderson 1898; Marx, et al., 2009; Vidal et al., 2009; Zaher, et al., 2009; 1958 and 1968; Werner, 1983; Goodman and Pyron, et al., 2011) to resolve the cladistic Hobbs, 1994). The suborder Serpents is distributed all relationships among snakes and to clarify their over the world (McDowell, 1987; Zug et al., 2001). phylogeny. Major changes to colubroid taxonomy have The Superfamily Colubroidea represents nearly 2500 been proposed based on molecular studies (Lawson et species of extant snakes (Gasperetti, 1988; Pough et al., 2005; Burbrink et al., 2007; Zaher et al., 2009). al., 2004). The monophyletic Colubroidea Snakes Also, the molecular RAPD-PCR technique has been species were established primarily on basis of external used as an important tool in genetic studies of snakes and taxonomical features (Zaher, 1999). Also, the (Prior et al., 1997; Jaggi et al., 2000; Dutra, et al., herpetological studies are subdivided Colubroidea into 2008). the families Viperidae, Elapidae, Atractaspididae, Although their morphology has been investigated Colubridae (Pough et al., 2004), while Dowling and previously, there are still major gaps in our knowledge Jenner (1988) reserved the superfamily Colubroidea to of the relationships of these animals. These gaps may the families Colubridae and Natricidae. Morever, hide important differences between ancient taxonomies Zaher et al. (2009) classified the family and molecular phylogenies which yet, the few species Psammophidae within the superfamily Elapoidea and and genera were included in these phylogenies , the families Colubridae and Natricidae was kept within leaving the classification of many genera in question the superfamily Colubroidea. Also, colubroids have (Lawson et al., 2005; Kelly et al., 2009; Zaher et al., been studied in historical biogeography (Pyron and 2009). Burbrink, 2009; Daza et al., 2010). Previous The family Colubridae is the most diverse, descriptions of the external and taxonomical features of widespread, and contains greater than 1800 species some snakes have been ambiguous and unreliable. within all of Serpents (Pough et al., 2004). Goodman Therefore, several authors used the karyological and Hobbs (1994) have been recorded the distribution 423 Life Science Journal, 2012;9(1) http://www.lifesciencesite.com of colubrid species of the family colubridae in the form with a slightly less slender body, weakly striped northern portion of the Egyptian Eastern Desert. These pattern and greyish belly (Bons and Geniez 1996; include: Coluber florulentus, C. rhodorhachis, C. Rato, et al., 2007). The occurrence of striped and rogersi, Lytorhynchus diadema, Malpolon moilensis, unicoloured morphotypes has also been recorded in Psammophis schoknri, P. aegyptius, and Spalerosophis Israel and Sinai (Kark et al. 1997; Rato et al., 2007). diadema. There are areas within Egypt where P. Broadley (1977, 2002) involved the species schokari, aegyptius and P. schokari are sympatric and both have aegyptius, punctulatus, elegans and trigrammus in the been collected in the Egyptian Eastern Desert ‘‘Psammophis schokari group”. Kelly et al. (2008) (Goodman et al., 1985). The classification of this noted that the P. cf. sibilans (Ethiopian), P. rukwae, P. group into subfamilies remain dissenting Topics subtaeniatus, P. sudanensis and P. orientalis are (McDowell, 1987; Vidal and Hedges, 2002; Kelly et involved in the one group. The RAPD technique has al., 2003; Nagy et al., 2003). The Colubridae comprise been used as a important tool in genetic studies of 12 subfamilies, Xenodermatinae, Pareatinae, snakes (Prior et al., 1997; Jaggi et al., 2000; Dutra, Calamariinae, Homalopsinae, Boodontinae, et al., 2008). Also, Broadley (1977) reported data of Pseudoxyrhophiinae, Colubrinae, Pseudoxenodontinae, Psammophis sibilans. Natricinae, Dipsadinae, and Xenodontinae (Zaher, This investigation aimed to illustrate the genetic 1999). The monophyly of the subfamilies Colubrinae, diversity between some common Egyptian colubrid Natricinae, Psammophiinae, and Xenodontinae appears snakes of the family Colubridae by using RAPD- PCR to be common to several molecular studies (Cadle, technique. 1988; Dowling et al., 1996; Gravlund, 2001; Kelly et al., 2003). While, Dowling and Jenner (1988) 2. Materials and Methods reserved the subfamily Colubrinae and Natricinae Animal dealer collected eight Egyptian colubrid within the families Colubridae and Natricidae, species (snakes) from different localities of Egypt. The respectively. Family Colubridae is now represented by eight species are belonging to four genera. twelve genera (Dolichophis, Eirenis, Hemorrhois, Morphological identification and classification of the Lytorhynchus, Malpolon, Natrix, Platyceps, animals as well as scientific and common names of Psammophis, Rhagerhis, Rhynchocalamus, these species was carried out according to previous Spalerosophis and Telescopus) including 24 species works (Anderson, 1898; Marx, 1968; Goodman and (Amr and Disi, 2011). Hobbs, 1994). The studied species are present in Table The molecular phylogenetic relationships of the 1. colubrid species (subfamily Colubrinae) were recorded by several authors (Lawson et al., 2005; Gravlund, Genomic DNA extraction 2001; Kelly et al., 2003). Muscle tissue from the snakes were taken and Kelly, et al (2008) found that the family stored at -20 oc. DNA extracted according to the Psammophiidae includes eight genera and about 50 method of Sambrook (1989) with slight modifications. species. Phylogenetic studies of the family DNA quality and concentration determined by Psammophiidae has been establish based on spectrophotometric analysis and run in 0.7 % agarose immunological data (Cadle, 1994) and mitochondrial gel. Each sample of DNA was examined by optical DNA sequences (Gravlund, 2001; Vidal and Hedges, density values at 260 and 280 nm. Optical density 2002; Nagy et al., 2003; Kelly et al., 2008). The ratios evaluated and only good quality DNA samples generic diagnosis for Psammophis carried out by were used in PCR. Broadley (2002) and Kelly et al. (2008). Bons and Geniez (1996) found that the genus Psammophis of the RAPD-PCR reaction subfamily Psammophiinae includes 24 species, most of 15 primers from Kits OP-B , OP-E and OP-O them with an African origin, but some also occur in the (Operon Technologies, Alameda, CA, USA) used for Middle East and Asia. Psammophis schokari is RAPD-PCR analysis (OPB-01, OPB-05, OPB-09, OP- widespread in North Africa having a Saharo-Sindian 10, OPB-12, OPB-13, OP-B14, OPB-17, OPB-19, OP- distribution; it is also found in the Middle East, Arabia, B20, OPE-01, OPE-05 OPE-10, OPO-01 and OPO- Iran, a large part of Afghanistan, Uzbekistan and 03). Only 10 primers (OPB-01, OPB-09, OPB-12, northwest India