B. KUTRUP, U. BÜLBÜL

Turk J Zool 2011; 35(4): 579-584 © TÜBİTAK Research Article doi:10.3906/zoo-0904-6

Comparison of skeletal muscle protein bands and trunk vertebrae count between ophryticus nesterovi and O. o. ophryticus populations in Turkey

Bilal KUTRUP, Ufuk BÜLBÜL* Department of Biology, Faculty of Arts and Sciences, Karadeniz Technical University, 61080 Trabzon - TURKEY

Received: 13.04.2009

Abstract: Skeletal muscle was investigated by SDS-PAGE protein electrophoresis for western versus eastern Turkish populations of the . Th e number of protein bands resolved was 25 in the west and 26 in the east. Th e modal number of trunk vertebrae was also compared among 10 populations of the eastern group (Artvin, Trabzon, Giresun, and Ordu) and 3 populations of the western group (Zonguldak, Bartın, and Karabük). Th e eastern group had 13 vertebrae except Şinik (Trabzon) specimens, which had 14 vertebrae, whereas the western group had 12 vertebrae. Th ese data supported the recognition of 2 taxa that would correspond to O. o. nesterovi in the west O. o. ophryticus in the east.

Key words: Skeletal muscle, SDS-PAGE, Ommatotriton ophryticus, protein band

Türkiye’deki Ommatotriton ophryticus nesterovi ve O. o. ophryticus populasyonları arasında iskelet kası protein bantlarının ve gövde omuru sayılarının karşılaştırılması

Özet: Ommatotriton ophryticus türünün Türkiye’deki doğu ve batı populasyonlarında iskelet kası SDS-PAGE protein elektroforezi vasıtasıyla incelenmiştir. Protein bandı sayısı batıda 25, doğuda 26 olarak bulunmuştur. Ayrıca doğu grubuna ait on (Artvin, Trabzon, Ordu ve Giresun) ve batı grubuna ait üç populasyon (Zonguldak, Bartın ve Karabük) arasında gövde omuru sayıları karşılaştırılmıştır. Doğu grubu örnekleri, 14 omura sahip Şinik (Trabzon) örnekleri haricinde 13 omura sahip olarak bulunurken, batıdaki örneklerin omur sayısı ise 12 olarak bulunmuştur. Bu veriler batıda O. o. nesterovi, doğuda O. o. ophryticus’a karşılık gelen iki takson tanımlamasını desteklemektedir.

Anahtar sözcükler: İskelet kası, SDS-PAGE, Ommatotriton ophryticus, protein bandı

Introduction Jordan (Borkin et al., 2003; Litvinchuk et al., 2005b; Th e banded newt, vittatus (Jenyns, 1835), Pearlson and Degani, 2007). For the taxonomical is distributed throughout the western Caucasus, status of banded newt, Triturus vittatus (Jenyns, Turkey, Lebanon, Syria, Israel, Iraq, and perhaps, 1835), it was suggested before that 3 subspecies, T. v.

* E-mail: [email protected]

579 Comparison of skeletal muscle protein bands and trunk vertebrae count between Ommatotriton ophryticus nesterovi and O. o. ophryticus populations in Turkey vittatus (Jenyns, 1835), T. v. cilicensis (Wolterstorff , Materials and methods 1906), and T. v. ophryticus (Berthold, 1846), are Collecting samples distributed in Turkey (Schmidtler and Schmidtler, Totally 203 adult (106 females and 97 males) O. 1967; Atatür, 1974; Özeti and Yılmaz, 1994; Arntzen ophryticus specimens (24 specimens from the western and Olgun, 2000; Borkin et al., 2003). In contrast, Black Sea and 179 specimens from the eastern Black Steinitz (1965) reported that only T. v. vittatus Sea) were collected from 21 diff erent populations. subspecies is distributed in Southern Anatolia and From the collected specimens, 107 individuals were there are 2 subspecies of T. vittatus (T. v. vittatus and used to determine trunk vertebrae count and 96 T. v. ophryticus) in Turkey. Th e species of the genus individiuals were used for SDS-PAGE. O. ophryticus Triturus are divided into 2 groups: the large-bodied specimens were collected from April to June in 2005. and the small bodied newts. Although the Th e specimens were caught by netting from their banded newt, T. vittatus, is intermediate in size, it breeding ponds. is considered to be more closely related to the large- Trunk vertebrae count bodied group. Recently, Litvinchuk et al. (2005a) Th e number of trunk vertebrae was determined proposed the Ommatotriton genus name for the for 93 specimens from 10 populations belonging to banded newt. On the other hand, Litvinchuk et al. the eastern group (Artvin, Trabzon, Giresun, and (2005a) also reported that T. vittatus is proposed to Ordu) and for 14 specimens from 3 populations have 2 species (O. ophryticus and O. vittatus) in Turkey belonging to the western group (Zonguldak, Karabük, in terms of their trunk vertebrae count, genome size, and Bartın) (Figure 1) by dissecting of dorsal muscle and allozyme data. According to their fi ndings, the according to Litvinchuk et al. (2005b). northern taxon, O. ophryticus, is subdivided into 2 SDS-PAGE geographic fragments: the western group is allocated to the O. ophryticus nesterovi subspecies while the In SDS-PAGE experiments, totally 96 adult (46 females and 50 males) O. ophryticus specimens eastern group is named O. ophryticus ophryticus. (12 specimens from the western Black Sea and Smith et al. (2008) studied CO1 DNA barcoding 84 specimens from the eastern Black Sea) were and they stated that the 2 species were used from 10 populations, (6 ♀♀ and 6 ♂♂ from shown to be deeply divergent. Ommatotriton vittatus Zonguldak (Kilimli), 4 ♀♀ and 6 ♂♂ from Giresun was considered to represent 2 allopatric species – (Tamdere), 5 ♀♀ and 4 ♂♂ from Rize (Gündoğdu), O. vittatus in the southern and O. ophryticus in the 31 ♀♀ and 34 ♂♂ from Trabzon (Akçaabat 3 ♀♀ northern part of the range (Litvinchuk et al., 2005a) – and 4 ♂♂; Çamburnu 5 ♀♀ and 4 ♂♂; Hıdırnebi 4 with 4 subspecies of variable morphology. Similarly, ♀♀ and 4 ♂♂; Kirazlık 3 ♀♀ and 6 ♂♂; Maçka 5 ♀♀ Arntzen and Olgun (2000), Litvinchuk et al. (2001), and 6 ♂♂; Uzungöl 7 ♀♀ and 6 ♂♂; Gürbulak 4 ♀♀ and Litvinchuk et al. (2005b) mentioned that trunk and 4 ♂♂) in Turkey (Figure 1). In each SDS-PAGE vertebrae count and genome sizes were diff erent in experiment 1 specimen was used for each population the western and eastern populations of O. ophryticus and the experiments were repeated for all specimens in Turkey. in each population. Skeletal muscle protein samples of the newts were obtained by grinding 0.1 g of skeletal To test the suggestion made by Litvinchuk et al. muscles of each specimen in liquid nitrogen and by (2005a) that O. ophryticus could be represented by 2 adding 0.1 mL of double distilled water (DDW) and diff erent subspecies in northern Turkey, the present 0.2 mL of 2 × SDS gel-loading buff er (100 mM Tris- study aimed i) to compare skeletal muscle proteins Base pH 6.8, 4% SDS electrophoresis grade, 0.2% of the O. o. nesterovi from one western Black Sea and bromophenol blue and 20% glycerol) (Sambrook et O. o. opryticus from 9 eastern Black Sea populations al., 1989). Samples were boiled for 2 min in the 2 × in Turkey for the fi rst time by SDS-PAGE, and ii) to SDS gel-loading buff er to denature the proteins prior compare trunk vertebrae count in 3 O. o. nesterovi to gel-loading (Lutz et al., 2001). Th e SDS-PAGE 99 and 10 O. o. ophryticus populations in Turkey. program was used for boiling in a thermal block. Th e

580 B. KUTRUP, U. BÜLBÜL

12345678910 42° Zonguldak Bartın Karabük A Artvin Ordu Giresun Trabzon

40° TURKEY B

38°

C

01020 km 36°

26° 28° 30° 32° 34° 36° 38° 40° 42° 44° Figure 1. Map showing the localities of the studied samples.

size of the minigels was 8.3 × 7.3 cm and the resolving gels were 12% (w/v) gradient. Th e 12% gradient gels were prepared by putting 3.3 mL of DDW, 4 mL of 30% acrylamide mix (29.2% acrylamide and 0.8% N,N’-methylene-bis-acrylamide), 2.5 mL of 1.5 M Tris pH 8.8, 0.1 mL of 10% SDS, 0.1 mL of 10% ammonium persulfate, and 0.004 mL of TEMED into a beaker. Th e 5% stacking gels were prepared by putting 2.7 mL of DDW, 0.67 mL of 30% acrylamide mix, 0.5 mL of 1.5 M Tris pH 6.8, 0.04 mL of 10% SDS, 0.04 mL of ammonium persulfate, and 0.004 mL of TEMED into a beaker (Sambrook et al., 1989). For SDS-PAGE experiments, 1.85 μg of protein of each 1 μL sample was applied to the wells. Gels Figure 2. SDS-PAGE results of 1 Ommatotriton ophryticus nesterovi (from Zonguldak) and 9 O. o. ophryticus were run at a constant current of 20 mA for 60 min specimens; 100 μL (185 μg) of protein samples were (Lutz et al., 2001). Coomassie Brillant Blue (CBB) applied to the wells. An arrow was added to indicate R-250 was used to stain the gels. Th e gels were put the position of the lacking protein band of the O. O. into washing solution 1 (1 h), which was prepared by nesterovi specimen. Numbers from 1 to 10 represent mixing 50 mL of methanol, 10 mL of glacial acetic the names of the following populations : 1. Zonguldak, acid, and 40 mL of DDW for 60 min. Th en the gels 2. Uzungöl, 3. Hıdırnebi, 4. Akçaabat, 5. Giresun. 6. Rize, 7. Gürbulak, 8. Kirazlık, 9. Çamburnu, 10. were put into washing solution 2 (1 h), prepared with Maçka. 7 mL of glacial acetic acid, 5 mL of methanol, and 88 mL of DDW for 60 min (İnan, 2005). Finally the gels were scanned in a scanner and are shown in Figure 2. Appearances of the protein bands on the gels were Results clear and the bands were counted by 2 diff erent Figure 2 shows SDS-PAGE patterns of the skeletal researchers. Although the bands were clearly seen on muscle proteins in O. ophryticus. Total number of the gels, when the gels were scanned the appearances skeletal muscle protein bands was determined as 25 of the protein bands were not so clear and some of for the western Black Sea (Zonguldak) population vague bands appeared in Figure 2. An arrow was while there were 26 bands in the eastern Black Sea added to indicate the lacking protein band that populations. Th e lacking protein band for Zonguldak belonged to O. o. nesterovi specimen in Figure 2. specimens is shown in Figure 2. Aft er analyzing the

581 Comparison of skeletal muscle protein bands and trunk vertebrae count between Ommatotriton ophryticus nesterovi and O. o. ophryticus populations in Turkey data obtained from all specimens used in the SDS- species. Th e authors found that 16 protein bands were PAGE experiments, no diff erence was found between diagnostic for Channa gachua and Channa striatus males and females. Th e number of trunk vertebrae in while 10 and 15 bands were diagnostic for C. marulus O. ophryticus was 13-14 in the eastern group (Artvin, and C. punctatus by SDS-PAGE. Bülbül and Kutrup Trabzon, Giresun, and Ordu) while it was 12 in the (2007a) also stated that molecular weights of the western group (Zonguldak, Karabük, and Bartın). skeletal myosin, actin, troponin C, and tropomyosin Th e Table shows the trunk vertebrae count results. proteins diff ered between Xenopus tropicalis and Hyla japonica. In addition to these data, a detailed and systematic study of skeletal fi ber types in limb Discussion muscle of 2 frog species, Rana and Xenopus, by using In the present study, skeletal muscle protein myosin isoform-based criteria was performed by bands of the O. ophryticus samples from 1 western Rowlerson and Spurway (1988). Th ey found diff erent and 9 eastern Black Sea populations in Turkey were myosin isoforms in 2 diff erent frog species. Th is result compared by using SDS-PAGE. provided strong evidence that the diff erent fi ber types SDS-PAGE results showed that total number of in Rana and Xenopus limb muscle contained diff erent skeletal muscle protein bands was 25 in the western myosin heavy chains (MHC) isoforms. Th ese data Black Sea population while there were 26 bands in revealed that total number of skeletal muscle protein the 9 eastern Black Sea populations. Using the total bands and biochemical characteristics of the skeletal number skeletal muscle protein bands to compare muscle proteins could be diff erent among species or diff erent species or subspecies was also performed by subspecies. Bülbül and Kutrup (2007b). Th e results on the total In the present study, trunk vertebrae counts of number of protein bands of the green toad (Bufo the western and eastern Black Sea specimens of O. viridis) showed that Hatay specimens were similar to ophryticus were also compared. Th e number of trunk Kayseri, Rize, and Tekirdağ specimens, while Mersin vertebrae in O. o. ophryticus specimens was 13- specimens had 2 diff erent protein bands compared to 14 in the eastern group while it was 12 in the O. o. other specimens in their study. Similarly Hasnain et nesterovi specimens in the western group. Th is result al. (2005) studied the soluble muscle proteins in 4 fi sh is consistent with the result reported by Litvinchuk

Table. Trunk vertebrae counts of Ommatotriton ophryticus nesterovi and O. o. ophryticus specimens from Turkey.

Subspecies Name Locality n Voucher Number Trunk vertebrae count

O. o. nesterovi Karabük (Yenice) 2 (1♂, 1♀) TrUB-01-02 12 O. o. nesterovi Bartın (Kozcagız) 2 (1♂, 1♀) TrUB-03-04 12 O. o. nesterovi Zonguldak (Ereğli) 10 (5♂, 5♀) TrUB-05-14 12 O. o. ophryticus Trabzon (Gürbulak) 23 (5♂, 18♀) TrUB-15-37 13 O. o. ophryticus Trabzon (Hıdırnebi) 15 (7♂, 8♀) TrUB-38-52 13 O. o. ophryticus Trabzon (Beşirli) 12 (6♂, 6♀) TrUB-53-64 13 O. o. ophryticus Trabzon (Şinik) 9 (7♂, 2♀) TrUB-65-73 14 O. o. ophryticus Trabzon (Of) 8 (3♂, 5♀) TrUB-74-81 13 O. o. ophryticus Trabzon (Yalıncak) 2 (2♂) TrUB-82-83 13 O. o. ophryticus Trabzon (Sürmene) 2 (1♂, 1♀) TrUB-84-85 13 O. o. ophryticus Artvin (Erenköy) 11 (4♂, 7♀) TrUB-86-96 13 O. o. ophryticus Ordu (Ulubey) 4 (2♂, 2♀) TrUB-97-100 13 O. o. ophryticus Giresun (Kümbet) 7 (3♂, 4♀) TrUB-101-107 13

582 B. KUTRUP, U. BÜLBÜL

et al. (2005b). Th e modal number trunk vertebrae 1974; Özeti and Yılmaz, 1994; Arntzen and Olgun, count was 12-13 in the western group and 13-14 in 2000; Borkin et al., 2003). In agreement with our the eastern group in their study. Similarly, Arntzen results, Litvinchuk et al. (2005a) reported that the and Olgun (2000) also found that modal count was banded newt, T. vittatus, is represented by 2 species 13 in the western Black Sea samples while it was 14 (O. ophryticus and O. vittatus) in Turkey according in the eastern Black Sea samples. For other Urodelas, to their trunk vertebrae count, genome size, and Litvinchuk and Borkin (2003) reported that the allozyme data. Th ey proposed that the northern number of trunk vertebrae varied between 14 and taxon, O. ophryticus, is subdivided into 2 geographic 21 in the family Hynobiidae. Th e lowest number fragments: the western group is allocated to the O. o. was recorded in Ranodon sibiricus, whereas the nesterovi subspecies while the eastern group is named highest count was in Onychodactylus fi scheri. Th e O. o. ophryticus. Smith et al. (2008) studied CO1 DNA modal number of trunk vertebrae was 15 in Ranodon barcoding amphibians and they stated that the 2 sibiricus (and, probably, Hynobius nigrescens); 16 in species were shown to be deeply divergent. O. vittatus Hynobius leechii (and, probably, Hynobius nebulosus); was considered to represent 2 allopatric species – 17 in Salamandrella keyserlingii, Hynobius naevis, and O. vittatus in the southern and O. ophryticus in the Batrachuperus mustersi; and 18 in Onychodactylus northern part of the range (Litvinchuk et al., 2005a) japonicus. O. fi scheri was characterized by the higher – with 4 subspecies of variable morphology. Similar modal number, which was equal to 20. On the other to their proposal, we recommend that O. ophryticus hand, number of trunk vertebrae varies from 16 to 23 be subdivided into 2 geographic fragments as O. o. in slender , Batrachoseps, and variation nesterovi in western Black Sea and O. o. ophryticus in was found within populations, among populations, eastern Black Sea regions. and among species (Anderson, 1960; Wake, 1966; Th e skeletal muscle diff erences revealed by the Brame and Murrey, 1968; Yanev, 1978). Chan et al. present study for the O. ophryticus specimens needs (2001) stated that except for 1 individual with 14 to be supported by more comprehensive populations, trunk vertebrae all Cynops pyrrhogaster and Cynops especially for the western Black Sea specimens. cyanurus specimens had 13 trunk vertebrae. Th e results in the present study for O. ophryticus species showed that western Black Sea specimens Acknowledgements were diff erent from eastern Black Sea ones according Th is study was supported fi nancially by the to the total number of skeletal muscle protein bands Karadeniz Technical University Scientifi c Researches and trunk vertebrae counts contrary to the former Unit (2005.111.04.3). Language revision was common suggestion that 3 subspecies, T. v. vittatus, accomplished by Utku Avcı, who is a post-doctoral T. v. cilicensis, and T. v. ophryticus, are distributed in researcher at Georgia University in the United States Turkey (Schmidtler and Schmidtler, 1967; Atatür, of America.

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