BIHAREAN BIOLOGIST 12 (2): 65-69 ©Biharean Biologist, Oradea, Romania, 2018 Article No.: e171205 http://biozoojournals.ro/bihbiol/index.html

Phylogenetic relationships among the abdullahbayrami (Scorpiones: ) populations in Turkey inferred from mitochondrial DNA sequence data

Hüseyin AYHAN1,*, Erol ATAY2, Ahmet ÇARHAN3, Ersen Aydın YAĞMUR4, Özcan ÖZKAN5 and Bekir ÇELEBI6

1. Vocational High School of Health Services Ankara Yıldırım Beyazıt University, TR-06760 Çubuk, Ankara, Turkey. 2. Department of Biology, Faculty of Science and Arts, Mustafa Kemal University, TR-31060 Hatay, Turkey. 3. Medical Faculty, Yıldırım Beyazıt University, TR-06800Ankara, Turkey 4. Vocational High School Alaşehir, Celal Bayar University, TR-45600 Alaşehir, Manisa, Turkey. 5. Department of Biology, Faculty of Science and Arts, Karatekin University, TR- 18100 Çankırı, Turkey. 6. Turkish Public Health Institute, TR-06420 Ankara, Turkey. * Corresponding author, H. Ayhan, E-mail: [email protected]

Received: 29. March 2017 / Accepted: 23. June 2017 / Available online: 03. August 2017 / Printed: December 2018

Abstract. Mitochondrial DNA (mtDNA) has been widely employed in phylogenetic studies. was recognised has representing a different Leiurus in 2009, with several morphological characters that differentiate it from Leiurus quinquestriatus, the type species. In this study, Leiurus abdullahbayrami samples collected from the provinces of Hatay, Kilis, Gaziantep, Şanliurfa, Adiyaman, and Mardin in Turkey were used as study material. The genetic identification of the L. abdullahbayrami (Scorpiones: Buthidae) was carried out by using the 16S mitochondrial gene, subunit of mtDNA. According to the results of this study, the little genetic difference between the groups was confirmed and the taxonomic validity of Leiurus abdullahbayrami was confirmed with molecular data. The 16S rRNA sequence data showed that the genus Leiurus seems to be represented in Turkey by a single species, L. abdullahbayrami. Nucleotide variations found in the current work constitute the first descriptive report for L. abdullahbayrami.

Key words: Leiurus abdullahbayrami, scorpion, molecular phylogeny, genotype, Turkey.

Introduction

According to Fet et al. (2003) all scorpion species with pow- erful neurotoxins for mammals have been found in the Buthidae , thus making them medically significant. Buthidae species belonging to the genera Androctonus, Leiu- rus and have effective venom, are the cause of scorpionism which is medically significant in Turkey are be- ing investigated (Demirsoy et al. 2001, Ozkan et al. 2006; 2010). The species of the Leiurus genus from Buthidae family have widely spread in North Africa, the and the (Lowe et al.). Tulga (1960) reported the first Leiurus record from Adıyaman province in Turkey as Leiurus quinquestriatus. Until the work of Yağmur et al. in 2009, the only species of Leiurus found in turkey was called L. quinquestriatus. However, with this study of Yağmur et al.

(2009), the type of Leiurus which is now in turkey is revised Figure 1. The color variations among populations in Leiurus abdulla- as Leiurus abdullahbayrami. This new species had small varia- hbayrami: A. Hatay province, B. Şanlıurfa province, C. Adıyaman tions between regions (Figs 1-2). Leiurus abdullahbayrami is (Yağmur et al. 2009). one of the 12 species belonging to the Buthidae family in Turkey (Yağmur 2011). The of the species Leiurus are among the most venomous of all scorpion species (Fet et al. 2003, Ozkan et al. 2011). The scorpion Leiurus abdullahbay- rami has one of the most powerful toxins found in Turkey (Erdeş et al. 2014). It is of utmost importance to the scorpion species Leiurus abdullahbayrami systematically, genetically and biogeographically and highlight its venom in cases of scorpionism (Lowe, Yağmur & Kovařík, 2014). Phylogenetic studies can use mtDNA to reveal evolutionary relationships between species, and also differences within species, com- bined with population genetics. Different gene regions are Figure 2. The head shield of Leiurus abdullahbayrami: A. Female para- used to infer phylogenetic relation in scorpions and in an- type (Gökçay Village, Adıyaman Province) B. Male paratype other species. These include gene sequences of mitochon- (Yamaçoba Village, Gaziantep Province) (Yağmur et al. 2009). drial ribosomal DNA (rDNA): 16S rDNA, 12S rDNA and nuclear ribosomal DNA (rDNA): 28s rDNA (Prendini & Wheeler 2005). Protein coding DNA can also be used, for ex- Wheeler 2005). The present study aimed to determine the ample the cytochrome oxidase subunit I (COI) (Prendini & nucleotide sequence of the 16S rRNA mitochondrial gene of 66 H. Ayhan et al.

Table 1. Shows Leiurus abdullahbayrami samples subject to this study.

Sample Number GenBank Accession Sample Localities – GPS Nucleotide sequences and Province Number (WGS 84 type of coordinate system) Length (bp) A1 / G.Antep KU318424 Gaziantep Sarısalkım Village, 1 km East, 37°05′46.5″N, 37°16′51.3″E, 1029 m, 306 A2 / G.Antep KU318425 Gaziantep Sarısalkım Village, 1 km East, 37°05′46.5″N, 37°16′51.3″E, 1029 m, 310 A3 / G.Antep KU318426 Gaziantep Sarısalkım Village, 1 km East, 37°05′46.5″N, 37°16′51.3″E, 1029 m, 328 A4 / G.Antep KU318427 Gaziantep Sarısalkım Village, 1 km East, 37°05′46.5″N, 37°16′51.3″E, 1029 m, 297 B1 / Kilis KU318428 Kilis Küplüce Village, 2 km East, 36°44′92″N, 37°15′19″E, 624 m, 345 B2 / Kilis KU318429 Kilis Küplüce Village, 2 km East, 36°44′92″N, 37°15′19″E, 624 m, 341 C1 / Ş.Urfa KU318430 Şanlıurfa Harran, Şuayipşehir Village, 2 km Northeast, 36°52′36″N, 345 39°22′18″E, 506 m, C2 / Ş.Urfa KU318431 Şanlıurfa Harran, Şuayipşehir Village, 2 km Northeast, 36°52′36″N, 344 39°22′18″E, 506 m, C3 / Ş.Urfa KU318432 Şanlıurfa Harran, Şuayipşehir Village, 2 km Northeast, 36°52′36″N, 340 39°22′18″E, 506 m, C4 / Ş.Urfa KU318433 Şanlıurfa Harran, Şuayipşehir Village, 2 km Northeast, 36°52′36″N, 344 39°22′18″E, 506 m, D1 / Hatay KU318434 Hatay Çakıryiğit Village, 1 km West, 36°15′42″N, 36°36′22″E, 225 m 289 D2 / Hatay KU318435 Hatay Çakıryiğit Village, 1 km West, 36°15′42″N, 36°36′22″E, 225 m 331 E1 / Mardin KU318436 Mardin Eskikale Village, 2 km East, 37°18′23″N, 40°46′28″E, 944 m 335 F1 / Adıyaman KU318437 Adıyaman Kâhta, 37°56′49″N, 38°44′47″E, 1535 m 223

Figure 3. The localities of the studied samples of Leiurus abdullahbayrami in Turkey.

the Leiurus abdullahbayrami species and also, to test if there is plification cycles of one minute at 94°C for denaturation, one minute a genetic concordance with the colour variations found for at 48°C for annealing, 1.5 minutes at 72°C for extension), and 10 this species between regions (Figs 1 and 2). minutes of final extension at 72°C. Finally, at the Figure 5 amplifica- tion products were run in 1.5% agarose electrophoretic gel and the

PCR samples were purified from the agarose gel using GeneAll Ge-

nomic DNA Purification kit (GeneAll, Korea). In the case the reason Material and Methods to use the agarose gel is, to concentrate a sample so that all the reac-

tion product can be loaded into a single well of the gel. DNA se- Sampling quencing procedure was performed by forward and reverse PCR The scorpions (14 units) were collected by digging their burrows and primer used during PCR reaction. The purified samples were se- under the stones during day time as and by using UV lamps at night, quenced with BigDye®Terminator V.3.1 (Applied Biosystems, USA) and they kept in 75-96% ethanol. The localization and coordinates of and were run on ABI PRISM® 3100 Genetic Analysis device. Chro- the localities where the samples were collected in the provinces Ha- matograms showing DNA sequencing results were analyzed using tay, Kilis, Gaziantep, Adıyaman, Şanlıurfa, and Mardin, are given in FINCH TV program®, version 1.4.0 (Geospiza, Inc.; USA). The ob- Table 1 and Figure 3. tained DNA sequences were edited and aligned using Basic Local

Alignment Search Tool, and aligned using software [BLAST, version Molecular Method 2.3.0 (http://blast.ncbi.nlm.nih.gov)]. All new DNA sequences were The study was carried out on the mtDNA isolated from the leg mus- submitted to the GeneBank database provided by National Biotech- cle tissue of the scorpions preserved in 96% ethanol. DNA was ex- nology Information Center (Accession numbers: KU318424- tracted by using GeneAll genomic DNA extraction kit (Seoul, South KU318437). Maximum parsimony tree was formed as regards the Korea) and standard phenol/chloroform method (Sambrook, Fritschi distance of samples using the MEGA7 software (Sudhir et al. 2016). & Maniatis 1982). The extracted DNA samples were measured with Leiurus quinquestriatus (Gen Bank Acc. AY226174.2) from the Buthi- Nano spectrophotometer in OD260 nm, and were kept in -20°C for dae family was chosen as the out-group to rot the tree and the long term storage. The PCR procedure was carried out with the for- “Pairwise Distance” calculation was carried out to determine the mo- ward primer 5’-CGA TTT GAA CTC AGA TCA-3’ and reverse lecular systematic relation of the samples used in the study. primer 5’-GTG CAA AGG TAG CAT AAT CA-3’ (Alpha DNA, Can- ada) to amplify the region of mitochondrial DNA 16S rRNA (Gan- tenbein et al. 1999). The amplification procedure was performed us- ing 1.25IU Taq DNA polymerase (Promega, USA), 2mM MgCl2, 1μL Results dNTPs (10mM), 5 μL genomic DNA (100ng), and RNase free dis- tilled water, and the reaction was carried out in total of 50μl. The The scorpions collected from the field were primarily exam- thermal cycle program is denaturation for 5 minutes in 94°C, 45 am- ined under a stereo microscope for morphological studies Phylogenetic relationships among the Leiurus abdullahbayrami populations in Turkey 67 and all were found to belong to the species Leiurus abdulla- tree. In order to show the molecular systematic relationship hbayrami according to the morphological examination. Sam- among the samples within the group, the sequence data ples were grouped according to colour variation into four from genus Leiurus as out-group L. quinquestriatus (Fig. 4). groups. According to study of Yağmur et al. (2009), Leiurus abdullahbayrami is easily distinguished from other members of the genus by pedipalp chela and metasomal segments that are distinctly shorter than in L. quinquestriatus; metasomal segment V has large and rounded lobes; trichobothrium db on the constant finger of pedipalp is located between trichobothria est and esb (in L. quinquestriatus it is located be- tween et and est). Gaziantep and Kilis Population; The carapace and are brown. The : yellowish between the segments I to IV, the segment V of the metasoma is brownish black. Leg femur and tibia are yellow; other seg- ments are light yellow. The carapace is brown. The mesosoma is black or blackish brown, the metasoma: yellow- ish between the segments I to IV, the segment V of the me- tasoma is black. Leg femur and tibia are dark yellow; other segments are light yellow. Figure 4. The Maximum Parsimony tree of the Leiurus abdullahbay- Hatay Population; The carapace and mesosoma are rami samples analysed. L. quinquestriatus (Gen Bank Acc. blackish brown. The mesosoma is black or blackish brown, AY226174.2), was selected as out-group. the metasoma: redish yellowish between the I to IV seg- ments, the segment V of the metasoma is black. Leg femur Genetic distance and tibia are dark yellow; other segments are redish yellow. Genetic distances among Leiurus abdullahbayrami species and Şanlıurfa Population; The carapace is light yellow with the outgroup species, L. quinquestriatus, were calculated for greyish brown dots. The mesosoma is greyish yellow, the 16rRNA. The sequence distance was determined in the sam- metasoma: light yellowish between the I to IV segments, the ples subjected to this study in relation to available scorpions segment V of the metasoma is greyish brown. Leg segments of Leiurus quinquestriatus (Gen Bank Acc. AY226174, Fet et are yellow; other segments are dark yellow. al., 2003). The sequence distance we found is between 0,488 Adıyaman Population; The carapace is dark brown close and 0,512(%). The maximum interspecific distance was ob- to black and there is a semi-circle region anterior to the cara- served between E1 and C4. The distances between Leiurus pace which is yellowish brown. The mesosoma is black or abdullahbayrami species and the outgroup species ranged be- blackish brown, the metasoma: yellowish between the I to tween 48.8% and 51.2%. The maximum distances were ob- IV segments, the segment V of the metasoma is black. served between E1 and the outgroup (Table 2). Approximately 300 base pair around product of 16S rRNA mitochondrial DNA region were amplified for four- teen samples, listed in Table 1. All results are based on the Discussion pairwise analysis of fourteen sequences. The evolutionary distances were computed by using the maximum parsimony Recently, new scorpion species or subspecies have been (MP) method and are in the units of the number of base sub- identified based on molecular techniques (mtDNA) is com- stitutions per site. “Bootstrap” and “Interior Branch Length” monly used in phylogenetic and phylogeographic studies tests have used for the reliability of the formed phylogenetic (Gantenbein et al. 2005). Studies towards putting forward

Table 2. Genetic distance values of the DNA sequences (%).

68 H. Ayhan et al.

A and F were aligned each other, respectively. Similarly, 97%, 97, 94% and 96% similarity were obtained respectively when Group B and C, Group B and D, Group B and E, and Group B and F were aligned (Table 2). In another compari- son, 100%, 96% and 99% similarity were found respectively when Group C and D, group C and E and Group C and F were aligned. 96% and 99% similarity were acquired respec- tively when Group D and E and Group D and F were aligned. There was 94% similarity when Group E and F were aligned (Table 2). Comparisons at the nucleotide level to- wards 14 field sampled show that all the field sample repre- sent Leiurus abdullahbayrami and there is 96-100% similarity was found for the mtDNA 16S rRNA region. When the studied samples group A, B, C, D, E and F. were compared with the reference Leiurus quinquestriatus, 87%, 86%, 88%, 88%, 87% and 94% similarity was achieved, respectively. According to the results of this study, the little genetic difference between the groups was confirmed and the taxonomic validity of Leiurus abdullahbayrami was con- firmed with molecular data. The 16S rRNA sequence data showed that the genus Leiurus seems to be represented in Turkey by a single species, L. abdullahbayrami. The results ob- tained in this paper increased the genetic information and contribute to the work of evolutionary taxonomists who work with scorpions. We aim to use different genetic loci in Figure 5. PCR products used to agarose gel electrophoresis. future phylogenetic studies to confirm the current results and to enlighten the reason beneath different morphology.

In the future, the genetic variation and the composition of the taxonomic classification of scorpions and the relation be- scorpion venom should be investigated for the development tween phylogenetic still continue (Prendini et al. 2003, of more effective antivenom to be used in scorpion enveno- Soleglad & Fet 2004, Sharma et al. 2015). Along with the mation. The investigation of the diverse populations could taxonomic studies of the scorpions, scorpionism cases also help to understand the origin and current distribution of L. constitute an important study field. In this study, the mor- abdullahbayrami. In addition, the preliminary tree presented phological difference of Leiurus abdullahbayrami to Leiurus in this study may give an indication of the relationships quinquestriatus was observed again on the field collected within the L. abdullahbayrami species in the scorpion fauna of samples. The 16S rRNA sequence data, was revealed for the Turkey. first time for L. abdullahbayrami in this study and the ob- tained sequence data were compared to L. quinquestriatus. The background colour of prosoma, mesosoma and me- tasomal segment V of the new species is black or blackish- Acknowledgements. The authors wish to thank Alparslan CERİT brown (Fig. 1). Coloring is clearly variable: Hatay population and Murat AKYÜREK for their help during study activities. This has darker coloration on prosoma, mesosoma and me- study Dr. Hüseyin AYHAN's doctoral thesis was taken from the study and study was supported by Mustafa Kemal University tasomal segment V in Gaziantep, Kilis, Adıyaman and Kah- Scientific Research Projects Unit (project no: 13982). ramanmaraş populations; carapace in Hatay population has no yellow spots. On the other hand, background colour of prosoma, mesosoma and metasomal segment V in Şanlıurfa population is lighter than in Gaziantep, Kilis, Adıyaman and References

Kahramanmaraş populations (Fig. 1). Juveniles always have Demirsoy, A., Durmuş, Y., Akbulut, A. (2001): Türkiye scorpiones (akrep) darker coloration than adults in all populations. Therefore, a faunasının sistematik ve biyolojik yönden incelenmesi. Proje No: 1998 K great deal of variability in coloration is observed, perhabs 1001 40. Ankara: Çevre Bakanlığı Çevre Koruma Genel Müdürlüğü due to adaptation to local ecological and geographic condi- Hayvanları Koruma Dairesi Başkanlığı; 118 p. Erdeş, E., Doğan, T.S., Coşar, I., Danışman, T., Kunt, K.B., Şeker, T., Yücel, M., tions (Yağmur et al. 2009). The morphological difference of Özen, C. (2014): Characterization of Leiurus abdullahbayrami (Scorpiones: Leiurus abdullahbayrami from Leiurus quinquestriatus was sup- Buthidae) venom: peptide profile, cytotoxicity and antimicrobial activity. ported by the genetic data. Nucleotide variations found in The Journal of Venomous and Toxins including Tropical Diseases 20(1): 48. this study constitute the first descriptive report for L. abdul- Fet, V., Gantenbein, B., Gromov, A.V., Lowe, G., Lourenço, W. (2003): The first lahbayrami. BLAST analysis was carried out in order to com- molecular phylogeny of Buthidae (Scorpiones). Euscorpius 4(1): 1-10. pare the obtained DNA sequences of the relevant gene re- Gantenbein, B., Fet, V., Largiadèr, C., Scholl, A. (1999): First DNA phylogeny of the genus Euscorpius Thorell 1876 (Scorpiones: Euscorpiidae) and its bearing gion produced by PCR. This DNA sequence analyses for on the and biogeography of this genus. Biogeographica (Paris), Leiurus abdullahbayrami in this study showed 98%, 98%, 75(3): 59-72. 99%, 96% and 98% similarity when Group A and Group B, Gantenbein, B., Fet, V., Gantenbeın-Rıtter, I.A., Balloux, F. (2005): Evidence for recombination in scorpion mitochondrial DNA (Scorpiones: Buthidae). Group A and C, Group A and D, Group A and E, and Group Proceedings of the Royal Society Biological Sciences 272(1564): 697–704. Phylogenetic relationships among the Leiurus abdullahbayrami populations in Turkey 69

Lowe, G., Yağmur, E.A., Kovařík, F. (2014): A Review of the Genus Leiurus with morphological phylogenetic signal. Proceedings of the Royal Society B Ehrenberg, 1828 (Scorpiones: Buthidae) with Description of Four New 282: DOI: 10.1098/rspb.2014.2953. Species from the Arabian Peninsula. Euscorpius, No. 191. Soleglad, M.E., Fet, V. (2004): High-level systematics and phylogeny of the Ozkan, O., Adiguzel, S., Ates, C., Bozyigit, I., Filazi, A. (2006): Optimization of extant scorpions (Scorpions: Orthosteric). Euscorpius 11(1): 1-75. antiscorpion venom production. The Journal of Venomous Animals and Sudhir, K., Glen, S., Koichiro, T. (2016): MEGA7: Molecular Evolutionary Toxins including Tropical Diseases 12(3): 390-399. Genetics Analysis Version 7.0 for Bigger Datasets. Molecular Biology and Ozkan, O., Carhan A., Karaer, Z. (2010): A study on the genetic diversity of Evolution 33(7): 1870-1874. (Olivier, 1807; Scorpiones: Buthidae) from Turkey. Tulga, T. (1960): Türkiyede varlığı ilk defa tespit edilen bir akrep türü ( The Journal of Venomous Animals and Toxins including Tropical Diseases quinquestriatus) ile Prionurus crassicauda’ya karşı hazırladığımız akrep 16(4): 599-606. serumları arasında çapraz proteksiyon deneyleri (Cross-reactions between Ozkan, O., Yagmur, E.A., Ark, M. (2011): A newly described scorpion species, anti-scorpion (Buthus quinquestriatus) and anti-scorpion (Pri- onurus Leiurus abdullahbayrami (Scorpion: Buthidae), and the lethal potency and in crassicauda) sera). Türk Hijyen ve Deneysel Biyoloji Dergisi 20(2): 191–203. vivo effects of its venom. The Journal of Venomous Animals and Toxins [in Turkish] including Tropical Diseases 17(4): 414-421. Yağmur, E.A., Koç, H., Kunt, K.B. (2009): Description of a New Species of Prendini, L., Crowe, T.M., Wheeler, W.C. (2003): Systematics and biogeography Leiurus Ehrenberg, 1828 (Scorpiones: Buthidae) from Southеastеrn Turkey. of family Scorpionidae (: Scorpiones), with a discussion on Euscorpius, No. 85. phylogenetic methods. Invertebrate Systematics 17: 185-259. Yağmur, E.A. (2011): Güneydoğu Anadolu akrep (Arachnıda: Scorpiones) Prendini, L., Wheeler, W.C. (2005): Scorpion higher phylogeny and faunası: Sistematiği ve Zoocoğrafyası. Ege Üniversitesi, Fen Bilimleri classification, taxonomic anarchy, and standards for peer review in online Enstitüsü, Doktora Tezi. İzmir, 249s. [in Turkish] publishing. Cladistics 21(5): 446-494. Sambrook, J., Fritschi, E.F., Maniatis, T. (1982): Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory Press: 545 p. Sharma, P.P., Fernandez, R., Esposito, L.A., Gonzalez-Santillan, E., Monod, L. (2015): Phylogenomic resolution of scorpions reveals multilevel discordance