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Kotsakiozi Et Al. 2018 Molecular Phylogenetics and Evolution 125 (2018) 177–187 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Multilocus phylogeny and coalescent species delimitation in Kotschy's T gecko, Mediodactylus kotschyi: Hidden diversity and cryptic species Panayiota Kotsakiozia,b, Daniel Jablonskic, Çetin Ilgazd, Yusuf Kumlutaşd, Aziz Avcıe, Shai Meirif,g, Yuval Itescuf,g, Oleg Kukushkinh,i, Václav Gvoždíkj,k, Giovanni Scillitanil, Stephanos A. Roussosm,n, David Jandzikc,o, Panagiotis Kasapidisp, Petros Lymberakisa, ⁎ Nikos Poulakakisa,b, a Natural History Museum of Crete, School of Sciences and Engineering, University of Crete, Knosos Avenue, Irakleio 71409, Greece b Department of Biology, School of Sciences and Engineering, University of Crete, Vassilika Vouton, Irakleio 70013, Greece c Department of Zoology, Comenius University in Bratislava, Mlynská dolina, Ilkovičova 6, 84215 Bratislava, Slovakia d Dokuz Eylül University, Faculty of Science, Department of Biology, 35160 Buca/İzmir, Turkey e Adnan Menderes University, Faculty of Science and Arts, Department of Biology, 09010 Aydın, Turkey f School of Zoology, Tel Aviv University, 6997801 Tel Aviv, Israel g The Steinhardt Museum of Natural History, Tel Aviv University, 6997801 Tel Aviv, Israel h Department of Biodiversity Studies and Ecological Monitoring, T.I. Vyazemski Karadagh Research Station – Nature Reserve of Russian Academy of Sciences, Nauki Srt. 24, stm. Kurortnoe, Theodosia 298188, Republic of the Crimea, Russia i Department of Herpetology, Institute of Zoology of Russian Academy of Sciences, Universitetskaya Emb. 1, Saint Petersburg 199034, Russia j Institute of Vertebrate Biology of the Czech Academy of Sciences, 603 65 Brno, Czech Republic k National Museum, Department of Zoology, 193 00 Prague, Czech Republic l Department of Biology, Section of Animal and Environmental Biology, Laboratory of Histology and Comparative Anatomy, University of Bari Aldo Moro, via Orabona 4/ a, Bari, Italy m Department of Biological Sciences, Texas Tech University, MS 43131, Lubbock, TX, 79409-3131, USA n Department of Biological Sciences, University of North Texas, Union Circle 1155, 76203 Denton, TX, USA o Department of Zoology, Charles University in Prague, Viničná 7, Prague, 12844 Czech Republic p Hellenic Centre for Marine Research, Institute of Marine Biology, Biotechnology and Aquaculture, P.O. Box 2214, 71003 Irakleio, Greece ARTICLE INFO ABSTRACT Keywords: Kotschy's Gecko, Mediodactylus kotschyi, is a small gecko native to southeastern Europe and the Levant. It dis- Gekkonidae plays great morphological variation with a large number of morphologically recognized subspecies. However, it Divergence times has been suggested that it constitutes a species complex of several yet unrecognized species. In this study, we Species delimitation used multilocus sequence data (three mitochondrial and three nuclear gene fragments) to estimate the phylo- Species complex genetic relationships of 174 specimens from 129 sampling localities, covering a substantial part of the dis- Systematics tribution range of the species. Our results revealed high genetic diversity of M. kotschyi populations and con- Eastern Mediterranean tributed to our knowledge about the phylogenetic relationships and the estimation of the divergence times between them. Diversification within M. kotschyi began approximately 15 million years ago (Mya) in the Middle Miocene, whereas the diversification within most of the major clades have been occurred in the last 5 Mya. Species delimitation analysis suggests there exists five species within the complex, and we propose to tentatively recognize the following taxa as full species: M. kotschyi (mainland Balkans, most of Aegean islands, and Italy), M. orientalis (Levant, Cyprus, southern Anatolia, and south-eastern Aegean islands), M. danilewskii (Black Sea region and south-western Anatolia), M. bartoni (Crete), and M. oertzeni (southern Dodecanese Islands). This newly recognized diversity underlines the complex biogeographical history of the Eastern Mediterranean region. 1. Introduction southwestern and central Asia to northern India, western China, and southern Mongolia (Agarwal et al., 2014; Bauer et al., 2013). The The Palearctic “naked-toed geckos” include ∼100 species dis- taxonomy of these geckos has been unstable and remains unresolved, tributed from North Africa across the southern Balkans and because qualitative characters have been used for generic delimitation ⁎ Corresponding author at: Natural History Museum of Crete, School of Sciences and Engineering, University of Crete, Knosos Avenue, Irakleio 71409, Greece. E-mail address: [email protected] (N. Poulakakis). https://doi.org/10.1016/j.ympev.2018.03.022 Received 5 July 2017; Received in revised form 12 March 2018; Accepted 15 March 2018 Available online 16 March 2018 1055-7903/ © 2018 Elsevier Inc. All rights reserved. P. Kotsakiozi et al. Molecular Phylogenetics and Evolution 125 (2018) 177–187 Fig. 1. The study area and the sampling localities of the samples used in this study. Numbers correspond to the sampling localities codes given in Table S1. The map is redrawn based on the Mediodactylus kotschyi distribution according to Sindaco and Jeremčenko (2008) in order for the shaded area (A) to indicate the distribution range of the species. For clarity reasons, the specimens from the Aegean region and south continetntal Greece are presented in B. Populations of the different phylogenetic clades as presented in Fig. 2 are indicated with differently colored dots. Inset: Mediodactylus kotschyi (Vlorë, Albania); photo: Daniel Jablonski. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) (Bauer et al., 2013 and references therein). Indicative of the great with stony habitats at heights up to 1700 m a.s.l. (Böhme et al., 2009). confusion is that the species of the genus Mediodactylus were initially It is a small gecko distributed in the Eastern Mediterranean and Black assigned to the genus Gymnodactylus, later on to Cyrtodactylus and then Sea regions from southeastern Italy in the west to Turkish Transcau- removed to Tenuidactylus as a distinct subgenus (Szczerbak and casian provinces in the east, and from south Greece, Cyprus and Israel Golubev, 1984). At present, Mediodactylus has been recognized as a full in the south to the Danube River valley at the central and eastern monophyletic genus (Bauer et al., 2013; Macey et al., 2000). Balkans (in the limits of Serbia and Bulgaria) and Crimea in the north The genus Mediodactylus comprises 13 species (Uetz, 2018) with M. (Böhme et al., 2009; Fig. 1). It is considered introduced in Italy, Serbia kotschyi (Steindachner, 1870) being its type species. Mediodactylus and Hungary (Böhme et al., 2009; Urosevic et al., 2016). kotschyi is a thermophilic and xerophilic species inhabiting the Medi- Mediodactylus kotschyi displays great morphological variation which terranean and sub-Mediterranean landscapes and associated mainly is reflected in the at least 26 (Baran and Gruber, 1982; Szczerbak, 1960; 178 P. Kotsakiozi et al. Molecular Phylogenetics and Evolution 125 (2018) 177–187 Szczerbak and Golubev, 1986) described subspecies (but see also Uetz localities (Fig. 1) ranging from Italy to eastern Turkey and from Crimea (2018) who mentioned 30 subspecies), 17 of which are found in Greece to Israel covering almost the whole distribution range of the species (Valakos et al., 2008). The species shows very high inter-population (Sindaco and Jeremčenko, 2008). Exact sampling localities, specimen variability in external morphology (Beutler and Gruber, 1977; codes and GenBank accession numbers of all markers included in the Kukushkin and Sharygin, 2005), making morphological differentiation phylogenetic analyses are shown in Table S1. at the subspecific level difficult due to the overlap of morphological Total genomic DNA was isolated from tissue samples (tail or tongue) characters used in their taxonomy (Ajtić, 2014; Szczerbak and Golubev, of specimens that were either frozen (−80 °C) and/or preserved in 1986). The subspecies of M. kotschyi have been classified into four ethanol. DNA was isolated using an Ammonium Acetate based DNA groups (Beutler, 1981): (i.) the kotschyi group in Italy, Albania, FYROM, extraction procedure (Bruford et al., 1998). southwestern and central Bulgaria, mainland Greece, northern and Three mtDNA (COI, cyt b, 16S rRNA) and three nuDNA (RAG2, eastern Aegean islands, Cyclades islands, northern Sporades islands, MC1R, c-mos) markers were amplified. For the nuDNA dataset a subset Argosaronic islands, Ionian islands and Kythira; (ii.) the danilewskii of specimens that had been sequenced for the mtDNA markers, was group, which includes all the Asiatic subspecies and those from Crimea, sequenced for the three nuDNA markers. The samples for the nuDNA western Thrace (NE Greece), eastern Bulgaria and Cyprus; (iii.) the dataset were selected in order to represent all the major clades revealed bartoni group in Crete and its satellite islets, excluding Gavdos; and (iv.) by the phylogenetic analyses on mtDNA data based on the results of a the oertzeni group in the Kasos-Karpathos island group and Gavdos is- Poisson Tree Processes (PTP) analysis (for details see below). Thus, the land. A recent study, based on 18 morphological characters in M. kot- samples included in the nuDNA dataset and the concatenated mtDNA & schyi samples
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