Radka Musilová 1 , Vít Zavadil 2 & Petr Kotlík 3

1. Zamenis persicus Zamenis lineatus Russia Krasnodar (AY122699.1) Trans-Caucasian Clade Georgia Turkey Ararat (AJ277672) Czech Republic Poohří (n=9) Czech Republic Podyjí (n=7) Slovakia M.Karpaty 1 Slovakia M.Karpaty 2, 3 Slovakia M.Karpaty 4 Slovakia M.Karpaty 5 Slovakia M.Karpaty 6 Slovakia Vihorlat 1 (n=5) Eastern Clade Slovakia Vihorlat2 (n=2) Slovakia Čabraď Bulgaria 1 Bulgaria 2, 3 Serbia (n=2) Montenegro Germany Schlangenbad (n=5) Germany Burghausen 1 (n=5) Germany Hirschhorn (n=4) Germany Burghausen 2 Croatia Switzerland Jura Italy Rome (AY122696.1) France Villiers Western Clade France Loire (AJ277671) Italy Foresta Umbra 1 Italy Aviano Greece Italy/Slovenia Switzerland Olivone (AY122697.1) Italy Foresta Umbra 2 0.01 Fig. 3: Maximum likelihood phylogeny PHYLOGEOGRAPHY OF AN ISOLATED POPULATION OF THE AESCULAPIAN SNAKE (ZAMENIS LONGISSIMUS) Radka Musilová1, Vít Zavadil2& Petr Kotlík3 1) Czech University of Life Sciences Prague, Faculty of Environment, Kamýcká 129, Prague 6 - Suchdol, 165 21, e-mail: musilovaradka@fle.czu.cz 2) ENKI, Dukelská 145, 379 01 Třeboň, e-mail: arnoviza@atlas.cz 3) Institute of Animal Physiology and Genetics AS CR, Rumburská 89, Liběchov, 27721, e-mail: kotlik@iapg.cas.cz Introduction: The present isolated population of the Aesculapian Snake along the Ohře River valley in the western Czech Republic has long provoked debate over its origin (Mikátová, Zavadil 2001). Fossil records suggest that the scattered isolates in the Czech Republic, Germany and Poland, may represent fragmented relics of a wider distribution during warmer conditions now surviving under a sub-optimal climate (Fig. 1). Because snakes were kept in the temples built by the ancient Greeks and by the Romans in honor of their gods (Bodson 1981), an alternative hypothesis has been suggested that the isolated populations may represent descendants of escaped snakes conveyed to health resorts by the Romans, or even more recently by members of a noble family from Italy or France (Šolcová-Danihelková 1966, Haleš 1969). Fig. 1: Fossil records and isolated populations of Zamenis longissimus above the northern limit of the current continuous range (Böhme 1993, Ljungar 1995, Najbar 2000, Gomille 2002, Ivanov 2005) Methods: Total genomic DNA from specimens of Zamenis longissimus was extracted and then subjected to the polymerase chain reaction (PCR) with the primers for the mitochondrial genes cytochrome oxidase I (COI, 527 bp) and cytochrome b (Cyt b, 1117 bp). We compared these mitochondrial DNA sequences of 65 Aesculapian snakes from 27 localities throughout Europe and western Asia, including 5 published sequences (Lenk et al. 2001, Utiger et al. 2002). Aims: This study intends to contribute to clarifying the genetic and historical biogeographic relationships of the isolated Bohemian population of the Aesculapian Snake with other populations. Our understanding of its origin may provide important information for future management of the population. Results: Phylogenetic analyses identified three main phylogeographical lineages with distributions suggesting that they originated from separate glacial refugia that were most likely located in the western Europe, in the Balkans, and along the eastern Black Sea coast, respectively. Snakes from the Ohře valley were genetically close to snakes from the Carpathians and northern Balkans, and they were only distantly related to the snakes from western Europe, the Apennine peninsula and southern Balkans. German populations near Schlangenbad and Hirschhorn belongs to Eastern lineage as well and there is a contact of Western and Eastern Clade near Burghausen in Germany. Fig. 2: Distribution of main clades within the distribution range of Zamenis longissimus Conclusions: Our findings are consistent with the native origin of the Ohře population and suggest that it may represent a relic of a wider distribution that the eastern European lineage attained during warmer post-glacial conditions by colonization from the Balkan refugium. References: Bodson, L. 1981: L´Antiquité class., 50 (1/2): 57 – 78; Böhme, W. 1993: Äskulapnatter (Elaphe longissima Laurenti 1768). Handbuch der Reptilien und Amphibien Europas. Aula Verlag: 331 -372. Gomille, A. 2002: Die Äskulapnatter Elaphe longissima. Edition Chimaira, Frankfurt am Main, 158 pp. Edition Chimaira, 158 pp.; Haleš, J. 1969: Ochrana fauny, 3: 119 – 123; Ivanov, M. 2005: Přírodovědné studie Muzea Prostějovska, 8: 89-108; Lenk, P.et al. 2001: Amphibia-Reptilia, 22: 329 – 339.; Ljungar, L. 1995: Amphibia-Reptilia, 16: 93-94; Mikátová, B., Zavadil, V. 2001: Atlas rozšíření plazů v České republice. AOPK ČR: 113 – 123; Najbar, B. 2000: Bull. Pol. Ac. Biol., 48: 53-62; Šolcová – Danihelková, M. 1966: Sborník biol. geol. věd PF, 2: 183-187; Utiger, U. et al. 2002: Russian J. Herp. , 9: 105-124. Aknowledgements: We are grateful to many colleagues for providing samples. This work was supported by a FRVŠ grant (2558/2006), AOPK ČR and is a part of MŠMT č. 6293359101 and IRP IAPG grant no. AV0Z50450515.