Turkish Journal of Zoology Turk J Zool (2020) 44: 134-145 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-1911-14 Taxonomic investigation of the genus Ablepharus (Sauria; Scincidae) with molecular and morphological methods in Anatolian populations 1, 2 Emin BOZKURT *, Kurtuluş OLGUN 1 Department of Veterinary Sciences, Eldivan Vocational School of Health Services, Çankırı Karatekin University, Çankırı, Turkey 2 Department of Biology, Faculty of Science and Arts, Adnan Menderes University, Aydın, Turkey Received: 11.11.2019 Accepted/Published Online: 28.01.2020 Final Version: 04.03.2020 Abstract: Ablepharus Lichtenstein, 1823, which includes ten species, is distributed in eastern Europe and Asia. Four species are recorded in Turkey: A. kitaibelii, A. chernovi, A. bivittatus, and A. budaki. After molecular and morphological studies in Anatolia, the phylogenetic relationship of the genus is still very complicated. Here, we investigate the taxonomic status of Ablepharus in Anatolia using morphological and molecular methods. The genetic structure ofAblepharus populations in Anatolia was analyzed using both the nuclear (CMOS) and mitochondrial (cyt b and COI) gene regions. The maximum likelihood and Bayesian inference methods were applied for phylogenetic tree reconstruction, together with calculation of p-distances and reconstruction of haplotype networks. The morphological analysis was evaluated with SPSS by using 12 metric and 17 meristic characteristics. In total, 41 specimens and 279 specimens were used for the molecular and morphological analysis, respectively. A. bivittatus has highly different genetic and morphological characteristics compared to the others, and it shares similar morphological characteristics with the genus Asymblepharus, which are elliptical tympanum, hidden upper eye opening under 3 or 4 large shields, light and dark longitudinal stripes on the back, adpressed hind-limbs reaching to knees, and well-developed limbs with five toes. For this reason,A. bivittatus was placed in the genus Asymblepharus as Asymblepharus bivittatus comb. nov. A. budaki anatolicus was elevated to species level as A. anatolicus based on morphological and genetic data. In this study, Anatolian A. kitaibelii comprised two sister subclades, A. kitaibelii kitaibelii and A. kitaibelii stepaneki, and this research is the first record of A. kitaibelii stepaneki in Turkey. A. chernovi was divided into three subclades: A. chernovi chernovi, A. chernovi eiselti, and A. chernovi isaurensis. Key words: Skinks, Turkey, Asymblepharus, pholidosis, phylogeny 1. Introduction Asia and colonized the Balkan Peninsula from Anatolia Scincidae is the largest lizard family with 1598 species about 5.51 million years ago (Skourtanioti et al., 2016). representing more than 25% of the world’s lizard species Ablepharus kitaibelii (Bibron and Bory, 1833) was (Bauer, 1998; Hedges, 2014; Skourtanioti et al., 2016). This described from the Pylos ruins. In 1953, A. chernovi was family was divided into seven subfamilies by Hedges (2014): described by Darevsky in Armenia. Fuhn (1970) defined Acontinae, Egerniinae, Eugongylinae, Lygosominae, the subspecies of A. k. stepaneki and introduced five Mabuyinae, Sphenomorphinae, and Scincinae. Pyron subspecies: A. k. chernovi, A. k. fabichi, A. k. fitzingeri, A. et al. (2013) stated that Scincinae and Feylininae are k. kitaibelii, and A. k. stepaneki. Eremchenko and Sczerbak synonymous. Ten species belonging to Scincidae are (1986) considered A. chernovi as a separate species. distributed in Anatolia and these are Ablepharus bivittatus, Göçmen et al. (1996) described a new subspecies, A. A. budaki, A. chernovi, A. kitaibelii, Chalcides ocellatus, kitaibelii budaki, in Cyprus. Eumeces schneideri, Heremites auratus, H. septemtaeniatus, Schmidtler (1997) made a comprehensive revision H. vittatus, and Ophiomorus kardesi (Schmidtler, 1997; of the genus Ablepharus in Anatolia and the Middle East Ilgaz et al., 2007; Kumlutaş et al., 2007; Poulakakis et al., and (1) raised A. kitaibelii chernovi to the species level as 2008; Durmuş et al.; 2011; Baran et al., 2012; Karin et al., Ablepharus chernovi Darevsky, 1953 and recognized four 2016, Kornilios et al., 2018). subspecies (A. c. chernovi, A. c. eiselti, A. c. isaurensis, Ablepharus Lichtenstein, 1823 is distributed in eastern and A. c. ressli) in Anatolia; (2) raised A. k. budaki to the Europe and Asia and is currently represented by ten species level as Ablepharus budaki Göçmen et al. 1996 and species (Poulakakis et al., 2005). It originated in southeast described two subspecies (A. b. budaki and A. b. anatolicus) * Correspondence: [email protected] 134 This work is licensed under a Creative Commons Attribution 4.0 International License. BOZKURT and OLGUN / Turk J Zool in southern Anatolia; and (3) restricted the distribution The mitochondrial cyt b region was also amplified via of A. kitaibelii kitaibelii from the Balkan Peninsula to PCR using the following primers: PL 5’-AAC CAA GAC Central Anatolia. Skourtanioti et al. (2016) proposed that CTG TGA YAY GAA-3’ and PH 5’-GGC TTA CAA GAC the western Anatolian and Kastelorizo populations could CAR KGC TTT-3’. The PCR reaction was carried out in a represent a distinct species with the available name A. total volume of 25 µL, with the following conditions: an anatolicus. initial cycle of 94 °C for 3 min, followed by 35 cycles of 94 The aim of this study was to solve the taxonomic °C for 30 s, 52 °C for 40 s, and 72 °C for 90 s, and a final issues of the genus Ablepharus in Anatolian populations cycle of 72 °C for 10 min (Huang et al., 2013). In addition with morphological and molecular analyses. Here, to the previous gene region, the mitochondrial COI region two mitochondrial markers (cyt b and COI) and one was amplified via PCR using the RepCOI-F 5’-TNT TMT nuclear marker (CMOS) were used and a morphological CAA CNA ACC ACA AAG A-3’ and RepCOI-R 5’-ACT analysis was also performed using metric and meristic TCT GGR TGK CCA AAR AAT CA-3’ primers. The PCR characteristics. reaction was carried out in a total volume of 25 µL, with the following conditions: an initial cycle of 94 °C for 3 min, 2. Materials and methods followed by 40 cycles of 94 °C for 40 s, 49 °C for 30 s, and 2.1. Genetic methods 74 °C for 1 min, and a final cycle of 72 °C for 7 min (Nagy To analyze the molecular sequences, 41 samples (Figure et al., 2012). A commercial automated sequencer was used 1; Table 1) with total DNA extracted by using a standard to sequence the amplified products (GATC-Biotech AG, phenol/chloroform procedure (Hillis and Moritz, 1990) Konstanz, Germany). were used. The nuclear CMOS region was amplified via DNA sequences were aligned using the CLUSTAL W polymerase chain reaction (PCR) using the following algorithm as implemented in BioEdit version 7.2.5 (Hall, primers: G73.1 5’-GGC TRT AAA RCA RGT GAA GAA 1999) while manually adjusting the alignments. Two A-3’ and G74.1 5’-GAR CWT CCA AAG TCT CCA ATC- different phylogenetic approaches were used, maximum 3’. The PCR reaction was carried out in a total volume of likelihood (ML) and Bayesian inference (BI), for the 25 µL, with the following conditions: an initial cycle of 92 partitioned concatenated alignment. The best models of °C for 12 min, followed by 40 cycles of 94 °C for 1 min, 56 nucleotide substitution for each gene were calculated in Mr °C for 1 min, and 72 °C for 1 min, and a final cycle of 72 °C Modeltest v3.7 (Posada and Crandall, 1998) based on the for 5 min (Whiting et al., 2003). Akaike information criterion. The bootstrap method with Figure 1. Distribution of the lizards used in the molecular and morphological analysis. For more details on samples, check Table 1. Colors indicate the lineages based on the analysis (orange: A. bivittatus, claret red: A. chernovi chernovi, pink: A. chernovi eiselti, yellow: A. chernovi isaurensis, red: A. budaki budaki, green: A. budaki anatolicus, blue: A. kitaibelii kitaibelii, and turquoise: A. kitaibelii stepaneki). 135 BOZKURT and OLGUN / Turk J Zool Table 1. List of the materials used in the molecular and morphological analysis. Number of samples used in Working morphological GenBank accession number, cyt b/ Taxon Locality number analyses COI/CMOS (♂♂/♀♀/ juveniles) 1 0/1/0 A. c. isaurensis Between Gülnar and Mut, 18 km/Mersin MN447752/*/MN418805 2 1/2/0 A. c. isaurensis Meke Saline/Konya MN447755/MN447788/MN418805 3 0/1/0 A. c. isaurensis Alahan/Mersin MN447758/*/MN418805 4 0/0/1 A. c. isaurensis Berendi village/Karaman MN447762/MN447793/MN418805 5 1/1/0 A. c. isaurensis Ortaören/Mersin MN447757/MN447790/MN418805 6 0/3/0 A. c. isaurensis Beyşehir/Konya MN447765/*/MN418805 7 7/9/0 A. c. isaurensis Kohu Mountain/Antalya MN447761/MN447792/MN418808 8 0/1/0 A. c. eiselti Mansurlu/Adana MN447749/MN447785/MN418808 9 3/1/0 A. c. eiselti Çamlıyayla/Mersin MN447759/MN447791/MN418808 10 1/1/0 A. c. chernovi Evcihüyük village/Kahramanmaraş MN447748/*/MN418808 11 1/2/0 A. c. chernovi Akoluk village/Kayseri MN447751/*/MN418808 12 0/1/0 A. c. chernovi Suceyin village/Malatya MN447750/*/MN418808 13 0/1/0 A. c. chernovi Yumaklıcerit village/Adıyaman MN447753/MN447786/MN418808 14 0/0/2 A. c. chernovi Sarısalkım village/Gaziantep MN447756/MN447789/MN418808 15 1/1/0 A. c. chernovi Between Kilis and Hassa, 7 km/Kilis MN447760/*/MN418808 16 0/1/0 A. c. chernovi Milkisağ village/Tunceli MN447754/MN447787/MN418808 17 2/1/1 A. c. chernovi Akdamar Island/Van MN447763/*/MN418808 18 1/0/0 A. k. kitaibelii Osmaniye village/Aydın MN447764/*/MN418804 19 1/1/0 A. k. kitaibelii Eber Lake/Konya MN447766/MN447794/MN418804 20 4/6/2 A. k. kitaibelii Between Eğirdir and Gelendost/Isparta MN447773/*/MN418804 21 3/4/1 A. k. kitaibelii Eğirdir/Isparta MN447772/MN447796/MN418806 22 1/1/0 A.