© Firenze University Press www.fupress.com/ah Acta Herpetologica 5(2): 207-215, 2010 Electrophoretic comparison of blood-serum proteins of Apathya cappadocica (Sauria, Lacertidae) subspecies from Anatolia Çetin Ilgaz1, Hüseyin Arıkan2, Yusuf Kumlutaş3, Aziz Avcı4 ¹ Dokuz Eylül University, Fauna and Flora Research and Application Center, 35160, Buca-İzmir, Tur- key. Correspondig author. E-mail: cetin.ilgaz@deu.edu.tr ² Ege University, Faculty of Science, Department of Biology, Section of Zoology, 35100, Bornova-İzmir, Turkey E-mail: huseyin.arikan@ege.edu.tr 3 Dokuz Eylül University, Faculty of Education, Department of Biology, 35160, Buca-İzmir, Turkey. E-mail: yusuf.kumlutas@deu.edu.tr 4 Adnan Menderes University, Science and Art Faculty, Department of Biology, 09010, Aydın-Turkey. E-mail: aavci@adu.edu.tr Submitted on: 2010, 26th July; revised on: 2010, 26th August; accepted on: 2010, 8th October. Abstract. Blood-serum proteins of the known subspecies of Apathya cappadocica (Werner, 1902) were studied comparatively by polyacrylamide disc gel electrophoresis. In order to obtain useful biochemical data for classification, differences between the electrophoreograms of the samples included in the morphologically different subspe- cies were distinguished qualitatively and quantitatively. These comparisons indicated that electrophoretic results supported morphological discrimination of the known subspecies of A. cappadocica. Keywords. Apathya cappadocica, blood-serum proteins, Lacertidae, Anatolia. INTRODUCTION Apathya is a small genus of lacertid lizard including 2 species [Apathya cappadocica (Werner 1902) and Apathya yassujica (Nilson, Rastegar-Pouyani, Rastegar-Pouyani and Andrén 2003)] found in Central, East, South and Southeastern Anatolia, Northern Iraq, and West Iran (Eiselt, 1979; Baran and Atatür, 1998; Nilson et al., 2003; Arnold et al., 2007). A. cappadocica was first described as Lacerta cappadocica from Erciyes Mountain in Kayseri, Turkey (Werner, 1902). A. cappodocica is a polytypic species and includes five subspecies [A. c. cappadocica (Werner, 1902) – type locality: Erciyes Mountain, Turkey; A. c. urmiana (Lantz-Suchow, 1934) – type locality: 20 km SW of Rezaiyeh, Iran; A. c. wolteri (Bird, 1936) – type locality: 16 km W of Gaziantep, Turkey; A. c. muhtari (Eiselt, 1979) – type locality: 26 km SW of Bitlis, Turkey and A. c. schmidtlerorum (Eiselt, 1979) – type 208 Ç. Ilgaz et alii locality 10 km S of Diyarbakır, Turkey] (Lantz and Suchow, 1934; Bird, 1936; Eiselt, 1979; Baran and Atatür, 1998; Sindaco et al., 2000). After a detailed study by Eiselt (1979), regional studies on morphology and ecology of A. cappadocica in its distribution sites were conducted (Bischoff and Schmidtler, 1994; Schmidtler and Bischoff, 1995; Schmidtler, 1997; Anderson, 1999; Kumlutaş and Olgun, 1999). Schmidtler and Bischoff (1995) examined specimens separated into four groups collected in Gaziantep, Hatay, Adana and Mersin in terms of pholidolial characteristics. They found considerable differences between the specimens dependent on climatic con- ditions in terms of examined pholidolial characteristics. They finally stated that color- pattern features of the specimens belonging to three known subspecies A. c. wolteri, A. c. muhtari and A. c. schmidtlerorum show variability dependent on their habitats. The taxo- nomical status of known subspecies of A. cappadocica is doubtful. There was no informa- tion on blood serum electrophoretic pattern in the comparison between the known sub- species of A. cappadocica. In order to confirm the present taxonomical status of known subspecies of A. cappadocica, it is necessary to obtain further information outside of mor- phological studies and the purpose of the present study is to identify the similarities and differences of the patterns of blood serum proteins in known subspecies of A. cappadocica. MATERIALS AND METHODS Adult male and female specimens of A. cappadocica were collected from known distribution sites of each subspecies in the Southern and Southeastern part of Turkey between 10 June to 15 June 2005 by Y. Kumlutaş, Ç. Ilgaz and A. Avcı. The specimens were transferred to the laboratory to obtain blood samples for electrophoretic analysis. After obtaining blood samples they were fixed with 5% formaldehyde in 70% ethanol and then preserved in 70% ethanol according to the method described by Başoğlu and Baran (1977). Collection numbers (ZDEU, Zoology Department of Ege University) were given to the specimens kept in the Lab. of the Department of Biology at Buca Education Faculty of Dokuz Eylül University. Data on specimens used for electrophoretic analysis is given in Table 1. All blood-serum protein study specimens were of similar length, i.e. they were of similar age. Table 1. Data on the specimens of A. cappadocica used for electrophoretic analysis (N: Number of specimens) Subspecies N Locality Collection Date Altitude (m) Coordinates A. c. cappadocica 2 (1♂ - 1♀) Pozantı, Adana, Turkey 10.06.2005 1210 N 3733292 – E 3458453 A. c. wolteri 2 (1♂ - 1♀) Between Kilis and Hassa 32. km, Kilis, Turkey 11.06.2005 540 N 3650280 – E 3707517 A. c. urmiana 2 (1♂ - 1♀) Hasankeyf, Batman, Turkey 15.06.2005 479 N 3742742 – E 4124547 A. c. muhtari 2 (1♂ - 1♀) Küçükalanlı village, Şanlıurfa, Turkey 12.06.2005 799 N 3710524 – E 3838335 A. c. schmidtlerorum 2 (1♂ - 1♀) Between Diyarbakır and Siverek, Diyarbakır, Turkey 14.06.2006 1058 N 3750897 – E 3942794 209Comparison of blood-serum proteins of Apathya cappadocica For electrophoretic analysis, blood samples were obtained from two specimens (one male and one female) for each subspecies of A. cappadocica. Blood samples were obtained from the postor- bital sinuses of living specimens via heparinized hematocrit capillary tubes according to the method described by MacLean et al. (1973). Samples were centrifuged for 5 minutes at 600 g and were stored in equal amounts (4 μl) at -20°C for each separation until analysis. Blood-serum samples were sepa- rated using polyacrylamide-disc electrophoresis according to Davis (1964), slightly modified by Özeti and Atatur (1979). Electrophoretic separations were carried out at room temperature (20-25°C) with a Canalco Model 1200 electrophoresis apparatus. Separation gels were first stained with 0.5% Amido Black, and then de-stained passively with repeated 7% acetic acid baths. Gels were qualitatively evalu- ated directly from the electrophoretograms and densitometric tracing curves of the separations were obtained using a Gelman ACD-15 Model 39430 densitometer scanning at 500 nm. RESULTS All specimens examined were sexually mature and no obvious difference was record- ed in serum protein phenograms (in densitometric curves) between sexes in all subspe- cies. Consequently, the specimens were pooled by sex for further evaluation. We also pooled the same aliquots of serum together and so this situation is important for the den- sitometric quantitative analysis. Significant differences were established among the subspecies from the viewpoints of fraction numbers, electrophoretic mobilities and densities of the blood proteins which sug- gests that all subspecies of A. cappadocica are clearly distinct at the subspecific level (Fig. 1). Gel electrophoretograms of the blood protein samples of the five subspecies are shown in Fig. 1. The gel electrophoretograms of blood-serum proteins of a specimen from each subspecies, together with their densitometric tracing curves, are shown in Figs 2, 3, 4, 5 and respectively. In A. c. cappadocica, A. c. wolteri and A. c. urmiana the blood protein fractions were divided into 14 fractions or fraction groups (1 albumin-like fraction and 1 postalbumin- like fraction at albumin region and 12 globulin-like fractions at globulin region) while the total number of fractions or fraction groups were found to be 12 (2 albumin-like fractions and 1 postalbumin-like fraction at albumin region and 9 globulin-like fractions at globu- lin region) in A. c. schmidtlerorum. Finally the total blood protein fractions were divided into 11 fractions or fraction groups (1 albumin-like fraction and 1 postalbumin-like frac- tion at albumin region and 9 globulin-like fractions at globulin region) (Figure 2-6). Although all subspecies except A. c. schmidtlerorum show similarity in terms of albu- min fractions, the fractions matched to each other at the globulin region show both quali- tative and quantitative differences among all subspecies. DISCUSSION Many researchers have highlighted the taxonomic importance of the number of frac- tions, and the mobility and density of blood-serum proteins obtained from electrophoretic separation (Dessauer and Fox, 1956; Chen, 1967; Ferguson, 1980; Arıkan, 1990; Arıkan et 210 Ç. Ilgaz et alii al., 1998, 1999; Kumlutaş et al., 2007). Ferguson (1980) stated that while the quantitative difference of fractions could reflect gender, age, environmental and physiological factors, the qualitative differences of fractions could be caused by genetic variations. So, qualita- tive differences are important for taxonomic evaluations. Fig. 1. Blood protein samples electrophoretograms of the five subspecies of Apathya cappadocica. A: A. c. cappadocica, B: A. c. wolteri, C: A. c. urmiana, D: A. c. schmidtlerorum, E: A. c. muhtari. (S: Start, junction between the stacking and separation gels). Fig. 2. Gel photograph showing the electrophoretic separation of the blood protein sample obtained from A. c. cappadocica, together with its densitometric tracing curve. OD: Optical density, S: Start (junction between the stacking and separation gels), G1-G11: Globulins zone, PA: Postalbumine zone, A: Albumine zone. 211Comparison of blood-serum proteins of Apathya cappadocica Fig. 3. Gel photograph showing the electrophoretic separation of the blood protein sample obtained from A. c. wolteri, together with its densitometric tracing curve. For further explanation, see legend to Figure 2. Fig. 4. Gel photograph showing the electrophoretic separation of the blood protein sample obtained from A. c. urmiana, together with its densitometric tracing curve. OD: Optical density, S: Start (junction between the stacking and separation gels). 212 Ç. Ilgaz et alii Fig. 5. Gel photograph showing the electrophoretic separation of the blood protein sample obtained from A. c. schmidtlerorum, together with its densitometric tracing curve. OD: Optical density, S: Start (junction between the stacking and separation gels). Fig. 6. Gel photograph showing the electrophoretic separation of the blood protein sample obtained from A. c. muhtari, together with its densitometric tracing curve. OD: Optical density, S: Start (junction between the stacking and separation gels). 213Comparison of blood-serum proteins of Apathya cappadocica There is no information concerning its serological characterization on known A. cappadocica subspecies. The results obtained in the blood-serum electrophoretic sepa- ration indicate significant differences among known subspecies of A. cappadocica in terms of the number of fractions, and the mobility and density of blood-serum frac- tions. While A. c. cappadocica, A. c. wolteri and A. c. urmiana show significant differ- ences quantitatively, there is a considerable difference between A. c. schmidtlerorum and A. c. muhtari qualitatively. Finally, it should be stated that each subspecies has special electrophorenograms. The taxonomical status of this species, which has formed the subject of the study since 1902, the date when it was first identified, up to this day, has been continuous- ly controversial. The species which was identified as Lacerta cappadocica by Werner in 1902 was included in a new genus under the name of Apathya, taking these features into consideration: subdigital lamellae being obviously crytopodion, being black in color and having a semi-opal palpebral aperture that is generally composed of 6-8 scales in the lower eyelid; the existence of tiny postnasal plates in the lower part of the nos- trils; the first supraocular being multi partial; the existence of many small scales that form a transversal line on the posterior of anal; the occipital being wider than interpa- rietal; and the existence of pterygoid teeth (Mehely, 1907). Mehely’s Apathya genus was mentioned as the synonym of Latastia (Bedriaga, 1884) by Boulenger (1907). Later, this taxon was accepted in the world of science in the following century (Mertens, 1924). The taxon, which was evaluated under the species of Apathya until the mid 20th century, was included again in the species of Lacerta in the following decades (Mertens, 1952; Clark and Clark, 1973; Böhme, 1971). One genus which is evaluated in the Lacertidae family, but whose taxonomical condition has not been clearly highlighted, is Apathya (Mayer and Arribas, 2001). In the study which observed the morphological and molec- ular evaluation of 19 taxa in total, which are included in the Lacertini group in Pal- earctic – Oriental area; cappadocica form was regarded in Apathya (Arnold et al., 2007). In the study, as a result of both the evaluation of 64 different morphological characters and the observation of the mitochondrial DNA of taxa that formed the subject of the research with molecular techniques, the result was that Apathya has a close relationship with Hellonolacerta. In another study carried out recently (Pavlicev and Mayer, 2009), the result was contrary to the results obtained by Arnold et al. (2007); a clear interpreta- tion cannot be achieved regarding the taxonomical status of A. cappadocica and close relative forms of it (e.g., Timon lepidus and Lacerta agilis). As can be understood from the explanations given above, more studies need to be carried out at the molecular level together with morphological data in order to clarify the current taxonomical situation and relative relationship of Apathya more clearly. ACKNOWLEGEMENTS This work forms part of a Project (Project No. TBAG-104T017) supported by TÜBİTAK (The Scientific and Technical Research Council of Turkey). 214 Ç. Ilgaz et alii REFERENCES Anderson, S.C. (1999) The Lizard of Iran. Soc. for the Study of Amphibians and Reptiles, Oxford (OH). Arıkan, H. (1990): Rana ridibunda (Anura, Ranidae) populasyonları üzerinde morfolojik ve serolojik araştırmalar. Turk. J. Zool. 14: 40-83. Arıkan, H., Atatür, M.K., Mermer, A. (1998): An investigation on the blood serum pro- teins of Chalcides ocellatus (Sauria: Scincidae) populations from Southern Anatolia. Turk. J. Zool. 22: 175-177. Arıkan, H., Atatür, M.K., Çevik, İ.E., Kumlutaş, Y. (1999): A serological investigation of Lacerta viridis (Laurenti, 1768) (Sauria: Lacertidae) populations in Turkey. Turk. J. Zool. 23: 227-230. Arnold, E.N., Arribas, O., Carranza, S. (2007) Systematics of the palaearctic and oriental lizard tribe Lacertini (Squamata: Lacertidae: Lacertinae), with descriptions of eight new genera. Zootaxa 1430: 1-86. Baran, İ., Atatür, M.K. (1998): Turkish Herpetofauna (Amphibians & Reptiles). Republic of Turkish Ministry of Environment, Ankara, Turkey. Başoğlu, M., Baran, İ. (1977) Türkiye Sürüngenleri. Kısım 1. Kaplumbağa ve Kertenkele- ler. Ege Üniversitesi Fen Fakültesi Kitaplar Serisi, Bornova, İzmir, Turkey. Bird, C.G. (1936): The distribution of reptiles and amphibians in Asiatic Turkey, with notes on a collection from the vilayets of Adana, Gaziantep, and Malatya. Annals Magaz. Nat. Hist. 18: 257-281. Bischoff, W., Schmidtler, J.F. (1994): Ergebnisse zweier Lacertiden exkursionen nach Syr- ien. Die Eidechse 5: 4-22. Boulenger, G.A. (1907): Remarks on Prof. L. von Méhely’s paper ‘Zur Lösung der, Mura- lis-FrageV. Annals Magaz. Nat. Hist. 20: 39-46. Böhme, W. (1971): Über das Stachelepithel am Hemipenis lacertider Eidechsen und seine systematische Bedeutung. Zeits. Zoolog. Syst. Evolutionsforschung 9: 187-223. Chen, P.S. (1967): Separation of serum proteins in different amphibian species by poly- acrylamide gel electrophoresis. Experientia 23: 483-485. Clark, R.J., Clark, E.D. (1973): Report on a collection of amphibians and reptiles from Turkey. Occ. Papers Calif. Acad. Sci. 104: 1-62. Davis, B.J. (1964): Disc electrophoresis- II. Method and application to human serum pro- teins. Ann N. Acad. Sci. 121: 404-427. Dessauer, H.C., Fox, W. (1956): Characteristic patterns of orders of amphibia and reptilia. Science 124: 225-226. Eiselt, J. (1979): Ergebnisse zoologischer Sammelreisen in der Türkei Lacerta cappadocica Werner, 1902 (Lacertidae, Reptilia). Annal. Naturhist. Mus. Wien 82: 387-421. Ferguson, A. (1980): Biochemical Systematics and Evolution. Blackie. Glasgow and London. Kumlutaş, Y., Olgun, K. (1999): Ihlara Vadisi (Aksaray) Lacerta cappadocica Werner, 1902 (Sauria: Lacertidae) örnekleri hakkında. Turk. J. Zool. 23: 497-502. Kumlutaş, Y., Arıkan, H., Ilgaz, Ç., Kaska, Y. (2007): A new subspecies, Eumeces schneideri barani n. ssp (Reptilia: Scincidae) from Turkey. Zootaxa 1387: 27-38. Lantz, L.A., Suchow, G.F. (1934): Apathya cappadocica urmiana subsp. nov., eine neue Eidechsenform aus dem persischen Kurdistan. Zoolog. Anz. 106: 294-299. 215Comparison of blood-serum proteins of Apathya cappadocica MacLean, G.S., Lee, S.K., Wilson, K.F. (1973): A simple method of obtaining blood from lizards. Copeia 2: 338-339. Mayer, W., Arribas, O. (2001): Phylogenetic relationships of the European lacertid genera Archaeolacerta and Iberolacerta and their relationships to some other Archaeolac- ertae (sensu lato) from Near East, derived from mitochondrial DNA sequences. J. Zool. Syst. Evolut. Res. 41: 157-161. Mehely, L.V. (1907): Zur Lösung der ‘Muralis-Frage’. (Vorläufige Mitteilung). Annales His- torico-Naturales Musei Nationalis Hungarici 5: 84-88. Mertens, R. (1924): Amphibien und Reptilien aus dem nördlichen Mesopotamien. Abh. Ber. Mus. Magdeburg 3/5: 349-390. Mertens, R. (1952): Amphibien und Reptilien aus der Türkei. İstanbul Üniversitesi Fen Fakültesi Mecmuası 18: 41-75. Nilson, G., Rastegar-Pouyani, N., Rastegar-Pouyani, E., Andrén, C. (2003): Lacertas of South and Central Zagros Mountains, Iran, with descriptions of two new taxa. Russ. J. Herpetol. 10: 11-24. Özeti, N., Atatür, M.K. (1979): A preliminary survey of the serum proteins of a population of Mertensiella luschani finikensis Başoglu and Atatür from Finike in Southwestern Anatolia. İstanbul Üniversitesi Fen Fakültesi Mecmuası 44: 23-79. Pavlicev, M., Mayer, W. (2009): Fast radiation of the subfamily Lacertinae (Reptilia: Lacer- tidae): history or methodical artefact? Mol. Phylog. Evol. 52: 727-734. Schmidtler, J.F., Bischoff, W. (1995): Beziehungen zwischen lebensraum und morphologie bei Lacerta cappadocica WERNER, 1902 in der Türkei. Die Eidechse 6: 13-21. Schmidtler, J.F. (1997): Anmerkungen zur Lacertiden-fauna des südlichen Zentral Anatoli- en. Die Eidechse 8: 1-9. Sindaco, R., Venchi, A., Carpaneto, G.M., Bologna, M. (2000): The Reptiles of Anatolia: A checklist and Zoogeographical analysis. Biogeographia 21: 441-554. Werner, F. (1902): Die reptilien und amphibienfauna von Kleinasien. SB. Kaiserl. Akad. Wiss. Wien, Mathem.-Naturw. 111: 1057-1121.