Phylogenetic Relationship Among Five Geckos from Egypt

The Journal of Basic & Applied Zoology (2012) 65, 184–190

The Egyptian German Society for Zoology The Journal of Basic & Applied Zoology

www.egsz.org www.sciencedirect.com

Phylogenetic relationship among ﬁve geckos from Egypt based on RAPD-PCR and protein electrophoresis (SDS–PAGE)

Nadia H.M. Sayed

Zoology Dept., College for Women for Science, Arts and Education, Ain Shams University, Heliopolis, Cairo, Egypt

Received 28 February 2012; accepted 6 March 2012 Available online 24 September 2012

KEYWORDS Abstract Genetic variations between five gekkonid species from Egypt; Tropiocolotes tripolitanus, Gekkonidae; Tropiocolotes steudneri, Tropiocolotes nattereri, Tarentola mauritanica and Tarentola annularis were Protein electrophoresis; analyzed by SDS–PAGE for water soluble proteins and random amplified polymorphic DNA RAPD-PCR; (RAPD) analysis. Based on SDS–PAGE of water soluble proteins for all species, the obtained Phylogenetic relationship; results revealed a total of 17 bands at molecular weights that ranged from 95 to 16 kDa. The poly- Geckos morphic bands among species were 11 (64.7%) and the mean similarity matrix value between them was 70.7%. Using RAPD-PCR, the results showed eight total amplified bands at molecular weights that ranged from 1408 to 360 bp. The polymorphic bands between species were 7 (87.5%) and the mean similarity matrix between them was 44.6%. The dendrogram showed that, the five gekkonid species are separated from each other into two clusters. The first cluster contains three species of the genus Tropiocolotes. The second cluster includes the two species of the genus Tarentola. Based on SDS–PAGE and RAPD-PCR results, T. nattereri is sister to T. steudneri with higher genetic similarity than with T. tripolitanus. It is concluded that, the similarity coefficient and the genetic distance values between the five gekkonid species indicate that the five gekkonid species are not identical and are separated from each other. From these results, it is indicated that the protein and RAPD analysis are useful molecular tools to indicate genetic variation between the species in the same genus or in the different genera. ª 2012 The Egyptian German Society for Zoology. Production and hosting by Elsevier B.V. All rights reserved.

Introduction

The squamates are the most diversiﬁed group containing liz- E-mail address: [email protected] ards and snakes (Vidal and Hedges, 2009). Gekkonidae is Peer review under responsibility of The Egyptian German Society for one of the largest vertebrate group among squamates that Zoology. are distributed throughout the world (Vidal and Hedges, 2005). There are four subfamilies of the Gekkonidae (Diplo- dactylinae, Gekkoninae, Eublepharinae and Sphaerodactyli- Production and hosting by Elsevier nae) with 1130 species and 108 genera (Han et al., 2004).

2090-9896 ª 2012 The Egyptian German Society for Zoology. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jobaz.2012.07.004 Phylogenetic relationship among ﬁve geckos from Egypt based on RAPD-PCR and protein electrophoresis (SDS–PAGE) 185

Geckos have high diversity in arid and semiarid habitats in Africa and Australia (Baha El Din, 2006). Several investiga- tions have been recorded on the herpetofauna of reptiles in Egypt (Anderson, 1898; Marx, 1968; Goodman and Hobbs, 1994; Baha El Din, 2006). The phylogenic relationship among the species of the family Gekkonidae was based on morphological (Anderson, 1898; Marx, 1968), karyological (Kawai et al., 2009) and molecular studies (Joger, 1985; Rato et al., 2010; Busais and Joger, 2011). The genus Tarentola comprises about 20 morphologically similar species (Baha El Din, 1997; Harris et al., 2004a,b), which live mainly in semi-arid to arid habitats. They are distributed in North Africa, coastal regions of Mediterranean Sea, Macaronesian islands and also Cuba and the Bahamas. Tarentola mauritanica is distributed in Mediterranean regions (North Egypt) and extends across Northern Libya and South- ern Tunisia. The Tarentola annularis is distributed in a wide area across Africa, central Sudan, to the north along the Nile to the Nile Delta and Sinai Peninsula (Egypt) (Joger, 1984a; Figure 1 Gel electrophoresis represents protein bands from five Baha El Din, 2006). The phylogenetic studies of the genus Gekkonid species (lanes 1–5). M, protein marker with molecular Tarentola based on molecular analysis such as serum protein size (1 kDa). 1, Tropiocolotes steudneri;2,Tropiocolotes nattereri; electrophoresis, quantitative precipitin tests of serum albumin 3, Tropiocolotes tripolitanus;4,Tarentola mauritanica;5,Tarentola (Joger, 1984b, 1985), RAPD-PCR analysis (Ali, 2012), DNA annularis. sequences from mitochondrial and nuclear genes (Carranza et al., 2002; Jesus et al., 2002; Harris et al., 2004a,b; Perera and Harris, 2008) indicated that morphologically similar spe- Materials and methods cies exhibit genetic variation. The genus Tropiocolotes (Peters, 1880) comprises a group of Five gekkonid species from Egypt were collected from differ- small, nocturnal and ground dwelling geckos. Several authors ent localities (Table 1). The five species are belonging to two (Baha El Din, 2001; Rastegar-Pouyani et al., 2007; Wilms genera. Morphological identification and classification of ani- et al., 2010) studied the morphological characters of Tropiocol- mals as well as scientific and common names of these species otes steudneri, Tropiocolotes nattereri and Tropiocolotes tripo- were carried out according to previous works (Anderson, litanus. The distribution of Tropiocolotes ranges from the 1898; Marx, 1968; Goodman and Hobbs, 1994). Western Sahara across Northern Africa to Israel, Sinai, the Arabian Peninsula, Iran, eastern Afghanistan and Pakistan (Anderson, 1898; Rastegar-Pouyani et al., 2007). Molecular Protein electrophoresis (SDS–PAGE) studies on the genus Tropiocolotes (Ali, 2012) are very scarce. Molecular markers proven by analysis of proteins and Protein electrophoresis was carried out with sodium dodecyl RAPD-PCR have shown excellent potential to analysis of ge- sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). netic structure of germplasm for several varieties of organisms Muscles from the five geckos were taken and stored at (Studer, 1992). Sodium dodecyl sulfate–polyacrylamide gel 20 C. Muscles were grounded with 1 ml of 1· extraction electrophoresis (SDS–PAGE) is the most inexpensive, simple buffer; (10% SDS, 10 ml glycerol, 1 M Tris–HCl and 0.25 M and important tool that is widely used to differentiate the evo- EDTA, pH 8.8) and left overnight in refrigerator. The samples lution among the species (Ferguson, 1980; Avise, 1994; Mishra were centrifuged and the clear supernatants containing water- et al., 2010). The RAPD-PCR analysis is used to differentiate soluble proteins were used for electrophoresis. Muscle soluble the closely related species in order to determine the genetic protein fractions were separated exclusively on a vertical slab diversity between and within the species and is a useful method gel (19.8 cm · 26.8 cm · 0.3 cm) using the gel electrophoretic for showing of breeding populations (Welsh and Mc Clelland, apparatus (manufactured by APPEX) according to the method 1990; Williams et al., 1990, 1993). of Laemmli (1970). The final monomer concentration in the The present study was targeted to assess the genetic diver- 0.75 mm-thick slab gels was 12% (w/v) for the separating gel sity among five gekkonid species by using biochemical (pro- and 4% (w/v) for the stacking gel. Prior to loading, all samples tein) and molecular (RAPD-PCR) markers. were incubated in the presence of 1% (w/v) SDS and 100 mM

Table 1 Scientific name, common name and localities of five gekkonid species from Egypt. Scientific name Common name Locality Tropiocolotes steudneri (Peters, 1869) Steudner’s Gecko, Steudner’s Pigmy Gecko Sinai Tropiocolotes nattereri (Steindachner, 1901) Natterer’s Gecko, BorsTaht El Hagar Sinai Tropiocolotes tripolitanus (Peters, 1880) Tripoli Gecko, Tripoli pigmy Gecko, BorsTaht El Hagar Sinai Tarentola mauritanica (Linnaeus, 1758) Moorish Gecko, Moorish wall Gecko, Crocodile Gecko Abu Rawash Tarentola annularis (Geoffroy, 1809) Egyptian Gecko, white spotted Gecko, Bors Abu ArbaNoqat Abu Rawash 186 N.H.M. Sayed

Table 2 Binary data obtained for absence (0) and presence (1) from protein gel electrophoresis among ﬁve gekkonid species. BN RF MW Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 BF P 1 0.052 95 001000.2Unique 2 0.071 90 110000.4Polymorphic 3 0.108 83 111000.6Polymorphic 4 0.221 63 111111Monomorphic 5 0.247 59 010000.2Unique 6 0.260 57 000110.4Polymorphic 7 0.303 51 111111Monomorphic 8 0.334 48 111111Monomorphic 9 0.370 44 110000.4Polymorphic 10 0.394 41 000110.4Polymorphic 11 0.427 38 110000.4Polymorphic 12 0.451 36 111111Monomorphic 13 0.476 34 111000.6Polymorphic 14 0.522 30 110100.6Polymorphic 15 0.704 19 111111Monomorphic 16 0.739 18 000110.4Polymorphic 17 0.773 16 111111Monomorphic BN, band number; RF, relative front; MW, molecular weight in kilo Daltons (kDa); BF, band frequency; P, polymorphism. 1, Tropiocolotes steudneri;2,Tropiocolotes nattereri;3,Tropiocolotes tripolitanus;4,Tarentola mauritanica;5,Tarentola annularis.

et al. (1989) with slight modifications. DNA quality and con- Table 3 Total number of bands, polymorphic bands, % of centration were determined by spectrophotometric analysis polymorphic bands, mean band frequency, unique bands of five and run in 0.7% agarose gel. Each sample of DNA was exam- gekkonid species for water soluble protein. ined by optical density values at 260 and 280 nm. Optical den- Monomorphic bands 6 sity ratios were evaluated and only good quality DNA samples Polymorphic (without unique) 10 were used in PCR. Unique bands 2 Polymorphic (with unique) 11 RAPD-PCR Total number of bands 17 Polymorphism (%) 64.70% Three primers from Kits OP-D, (Operon Technologies, Ala- Mean of band frequency 0.624 meda, CA, USA) were used for RAPD-PCR analysis (OPD- 02, OPD-03 and OPD-05). Only one primer (OPD-05) with sequences D-05 50-TGAGCGGACA-30 showed some variations among five gekkonid species. RAPD-PCRs were carried out DTT for 5 min at 100 C. The samples were run at a constant as described by Williams et al. (1993). PCR cycles were per- voltage of 200 V applied for 45 min. The proteins were visual- formed with 60 s, 94 C for initial denaturation and then 35 cy- ized by Coomassie Brilliant Blue stain. cles of 20 s 94 C; 20 s 35 C; and 30 s 72 C. Final extension was performed at 72 C for 5 min. PCR amplifications were Genomic DNA extraction carried out in a 96-well Thermal cycler (Eppendorf Master Cy- cler) and all amplifications were carried out two times. A PCR Muscles from five geckos were taken and stored at 20 C. mixture without template DNA was placed in each analysis as DNA was extracted according to the method of Sambrook a control. The PCR products were separated on 1.5% agarose

Table 4 Similarity matrices (below) and genetic distances (above) among the ﬁve gekkonid species for water soluble protein data according to Nei and Li (1979). Tropiocolotes Tropiocolotes Tropiocolotes Tarentola Tarentola steudneri nattereri tripolitanus mauritanica annularis Tropiocolotes _ 0.04 0.24 0.36 0.43 steudneri Tropiocolotes 0.96 _ 0.27 0.39 0.45 nattereri Tropiocolotes 0.76 0.73 _ 0.37 0.33 tripolitanus Tarentola 0.64 0.61 0.63 _ 0.05 mauritanica Tarentola annularis 0.57 0.55 0.67 0.95 _ Phylogenetic relationship among ﬁve geckos from Egypt based on RAPD-PCR and protein electrophoresis (SDS–PAGE) 187

Table 6 Total number of bands, polymorphic bands, % of polymorphic bands, mean band frequency, unique bands from RAPD-PCR among ﬁve gekkonid species. Gel polymorphism Monomorphic bands 1 Polymorphic (without unique) 6 Unique bands 1 Figure 2 Dendrogram shows phylogenetic relationship among Polymorphic (with unique) 7 the ﬁve gekkonid species (1–5) based on protein data. Total number of bands 8 Polymorphism (%) 87.500% Mean of band frequency 0.475

RAPD banding patterns were scored for the presence (1) and absence (0) of bands for each sample. The statistical analysis of the data included the calculation of allele frequencies according to Nei (1987) and the number and percentage of polymorphic loci according to Nei (1973). Genetic similarity and genetic distance were estimated among species according to Nei and Li (1979). Based on the genetic similarity matrix, the species were clustered by the unweighted pair group method with arithmetic averaging (UPGMA) using the program NTSYS-pc version 2.1 (Rohlf, 2000).

Results

Muscle soluble protein (SDS–PAGE) analysis

Figure 3 Gel electrophoresis represents RAPD-PCR products Electrophoretic analysis was carried out on muscle soluble for DNA from five gekkonid species (lanes 1–5) with OPD-05 protein (SDS–PAGE) fraction of five gekkonid species. Gel primer. M, DNA marker with molecular size (1 bp ladder). 1, analysis of water soluble protein (SDS–PAGE) demonstrated Tropiocolotes steudneri;2,Tropiocolotes nattereri;3,Tropiocolotes 17 protein bands with different molecular weights that ranged tripolitanus;4,Tarentola mauritanica;5,Tarentola annularis. from 95 to 16 kDa in all genotypes (Fig. 1 and Table 2). The band frequencies ranged from 0.2 to 1.00 with a mean value equal to 0.624 (62.4%) and the unique bands are two bands. gels (Sigma) containing ethidium bromide in 0.5· TBE buffer All species shared 6 (35.3%) common bands and 11 (64.7%) at 100 V constant voltages. For evaluating the base pair length polymorphic bands (Table 3). The similarity matrix and the ge- of bands, DNA ladder (Fermentas) was loaded with each gel. netic distance were calculated according to the total number of protein fractions and the number of sharing bands between Data and statistical analysis species (Table 4). The similarity matrices ranged from 0.55 (55%) to 0.96 (96%) with an average of 0.707 (70.7%) and Protein and RAPD gels were documented using a digital cam- the genetic distances ranged from 0.04 (4%) to 0.45 (45%) with era (SONY, 5 MP) and on the basis of the band mobility. The an average of 0.293 (29.3%) between all species. T. steudneri is clear bands were scored using Totallab 120 Gel analysis pro- closer to T. nattereri (96%) than to T. tripolitanus (76%). gram (Nonlinear Inc., Durham, NC, USA). The protein and Moreover, the similarity coefficient between T. mauritanica

Table 5 Binary data obtained for absence (0) and presence (1) from RAPD gel electrophoresis among ﬁve gekkonid species. BN RF MW Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Frequency Polymorphism 1 0.365 1408 000100.2 Unique 2 0.424 1059 000110.4 Polymorphic 3 0.448 943 110000.4 Polymorphic 4 0.494 755 110000.4 Polymorphic 5 0.552 570 111111 Monomorphic 6 0.587 482 011000.4 Polymorphic 7 0.604 444 000110.4 Polymorphic 8 0.647 360 101100.6 Polymorphic BN, band number; RF, relative front; MW, molecular weight in kilo Daltons; BF, band frequency; P, polymorphism. 1, Tropiocolotes steudneri; 2, Tropiocolotes nattereri;3,Tropiocolotes tripolitanus;4,Tarentola mauritanica;5,Tarentola annularis. 188 N.H.M. Sayed

Table 7 Similarity matrices (below) and genetic distances (above) among the ﬁve gekkonid species for RAPD-PCR data according to Nei and Li, (1979). Tropiocolotes Tropiocolotes Tropiocolotes Tarentola Tarentola steudneri nattereri tripolitanus mauritanica annularis Tropiocolotes steudneri _ 0.25 0.43 0.56 0.71 Tropiocolotes nattereri 0.75 _ 0.50 0.78 0.71 Tropiocolotes 0.57 0.57 _ 0.50 0.67 tripolitanus Tarentola mauritanica 0.44 0.22 0.50 _ 0.25 Tarentola annularis 0.29 0.29 0.33 0.75 _

and T. annularis is 95%. The relationship between the five soluble proteins and RAPD-PCR analysis. The RAPD-PCR gekkonid species is presented in the form of a dendrogram revealed high polymorphic bands (87.5%) and low genetic sim- (Fig. 2). The dendrogram shows two main clusters, the first ilarity (44.6%) with high genetic distance (55.4%) among the cluster grouped Tarentola species while the other cluster is five gekkonid species. These results indicate that the five spe- formed by the other gekkonid species. T. nattereri is clustered cies are separated from each other. The present results are to T. steudneri in one clade and the two species are sister clade comparable to that found by Ali (2012) which found that the to T. tripolitanus. The present results indicated that the species similarity between the species is 42% by using a RAPD-PCR in the same genus are more related to each other (Table 4). technique. The UPGMA dendrogram (Fig. 4) and Table 7 show that, the species T. annularis and T. mauritanica are sis- Molecular genetic marker ters to each other where they have 75% genetic similarity and 25% genetic distance between them. Therefore, the two species Genomic DNA of the five gekkonid genotypes was extracted are closer to each other than the other studied species. Also, and used to execute RAPD-PCR. Three arbitrary oligonucleo- Ali (2012) found 57% genetic similarity between the two spe- tide primers produced multiple band profiles with a number of cies by using RAPD analysis. T. mauritanica species is charac- amplified DNA fragments. Only one primer (D5) reacted and terized by similar traditional morphology but shows high produced fragment with all genotypes (Fig. 3). The results of genetic diversity between the individuals within the same spe- the RAPD analysis are presented in Table 5. This primer cies (Carranza et al., 2002; Jesus et al., 2002; Harris et al., established eight different bands scored for the presence or ab- 2004a,b, 2009; Rato et al., 2010). Therefore, it is suggested that sence of bands at molecular weights that ranged from 1408 to T. mauritanica is classified into two subspecies, Tarentola mau- 360 bp among the five gekkonid species. The number of RAPD ritanica fascicularis and Tarentola mauritanica mauritanica bands ranged from 3 to 5 bands/species. Table 6 shows gel from the North African (Tunisia, Libya and Egypt). They polymorphism where seven bands (87.5%) were polymorphic. showed high genetic distinct polymorphism (8%) by using gene The band frequencies ranged from 0.2 to1.0 with a mean band sequences (Harris et al., 2004a,b, 2009). The mitochondrial ge- frequency of 0.475% (47.5) for all gekkonids and the unique netic distance between two morphologically similar species, T. band was one band. The similarity matrices among the five mauritanica and Tarentola deserti showed that despite some gekkonid species are presented in Table 7. The similarity coef- morphological similarity, the Tunisian and Libyan-Egyptian ficient values among the five gekkonid species ranged from T. deserti are not subspecies of T. mauritanica but Tarentola 22% to 75% with an average of 44.6%. The genetic distances fascicularis is subspecies of T. mauritanica (Joger and between the five gekkonids ranged from 25% to 78% with an Bshaenia, 2010; Rato et al., 2010). Bshaena (2011) found that average of 55.4%. The species of T. mauritanica and T. annu- T. m. fascicularis from Egypt formed a sister relationship with laris were closer to each other with 75% genetic similarity. T. T. sp. from Cyrenaica and T. a. annularis from Egypt formed a nattereri was nearer to T. steudneri (75%) than to T. tripolit- sister relationship with Tarentola boettgeri, from Canary anus (57%). Islands. In addition, they found intrageneric diversity and sys- The UPGMA dendrogram was constructed to show phylo- tematics in genus Tarentola in North Africa (Morocco, Alge- genetic relationships among the five gekkonid species based on ria, Tunisia, Libya and Egypt), in Saharan and coastal areas. genetic similarities (Fig. 4). It indicated that the five gekkonids The genus Tropiocolotes contains three species, T. steudneri, were clustered into two main groups. The first cluster grouped T. nattereri and T. tripolitanus. In the present study, the simi- T. nattereri, T. steudneri and T. tripolitanus. T. tripolitanus is larity coefficient between T. steudneri and T. nattereri is higher sister clade to the clade that contains T. nattereri and T. steud- (75%) than the similarity coefficient between them and T. tri- neri. The second cluster grouped the two species of the genus, politanus (57%). These results are similar to that presented by Tarentola. The T. steudneri and T. nattereri were clustered with Ali (2012) by using RAPD-PCR analysis who found that the Tarentola species. genetic similarity between T. nattereri and T. tripolitanus was 57.6%. Discussion Electrophoretic techniques for protein and isozyme polymorphism have been used as identification and quantitation In this study, the phylogenetic variation between five gekkonid methods and provide relationship between the changed expres- species T. nattereri, T. steudneri, T. tripolitanus, T. mauritanica sion of specific genes and changes in the environmental stress. and T. annularis were examined by using SDS–PAGE of water These changes in expression of genes would be involved in Phylogenetic relationship among five geckos from Egypt based on RAPD-PCR and protein electrophoresis (SDS–PAGE) 189

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