Phylogeography of the land snail genus Circassina (Gastropoda: Hygromiidae) implies multiple Pleistocene refugia in the western Caucasus region q Marco T. Neiber, Bernhard Hausdorf ⇑ Centre for Natural History, Zoological Museum, University of Hamburg, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany article info Article history: Received 4 March 2015 Revised 16 July 2015 Accepted 18 July 2015 Available online 26 July 2015 Keywords: Caucasus Colchis Dart apparatus Historical biogeography Phylogeography Pleistocene refugia abstract The phylogeny and historical biogeography of the Caucasian land snail genus Circassina was recon- structed using multilocus amplified fragment length polymorphism (AFLP) data and mitochondrial DNA sequences. Diversification within the group started with a divergence of populations from the west- ern Lesser Caucasus from those of the Greater Caucasus during the late Miocene. Distinct AFLP clusters and major mitochondrial clades separated by long internal branches lend evidence to the hypothesis of separate glacial refuges in the Lesser and Greater Caucasus during the Pleistocene. High genetic dis- tances across low geographic distances and admixture analysis revealed a phylogeographic boundary running through the Colchis lowlands, which may have been established and maintained in part by repeated transgressions of the Black Sea during the Pleistocene and Holocene. Localities in Ciscaucasia were probably colonised through long-distance dispersal across the main ridge of the Greater Caucasus. The phylogeny implies multiple independent losses of accessory genital organs, i.e. dart sac and mucus glands, within Circassina. None of the anatomically defined (sub-) species distinguished so far is monophyletic and there is gene flow between the two main population groups across the Colchis lowlands. Thus, we propose to classify these population groups as subspecies of a single species. Ó 2015 Elsevier Inc. All rights reserved. 1. Introduction The Caucasus region is ranked among the 25 most important global biodiversity hotspots (Myers et al., 2000; Zazanashvili et al., 2004). The importance of the western Caucasus, especially the Colchis region, as a glacial refugium where, among others, Neogene relict species survived as well as a centre of ongoing radi- ation has increasingly been appreciated (Hewitt, 2000; Pokryszko et al., 2011; Tarkhnishvili et al., 2012; Nakhutsrishvili, 2013; Tarkhnishvili, 2014; Walther et al., 2014). There is dissent, how- ever, on whether there existed a single continuous forest refugium at the eastern Black Sea coast as implied by some studies (van Andel and Tzedakis, 1996; Kikvidze and Ohsawa, 2001; Pokryszko et al., 2011; Tarkhnishvili et al., 2012; Volkova et al., 2013; Wielstra et al., 2013) or multiple forest refugia (Tarkhnishvili et al., 2000; Mumladze et al., 2013; Tarkhnishvili, 2014). Circassina Hesse, 1921 (Gastropoda: Hygromiidae) is a group of land snails endemic to the western and central Caucasus region and the eastern Pontus. It includes only two species, C. frutis (Pfeiffer, 1859) and C. lasistana Hausdorf, 2001. Abchasohela Hudec & Lezhawa, 1971, which was formerly classified as a sub- genus of Circassina (Hausdorf, 2001), proved to be more closely related to other hygromiid groups than to Circassina (Walther et al., in press). The Circassina frutis complex is exceptional among helicoid land snails because it is polymorphic with regard to the dart apparatus, i.e. the snails possess either a complete dart appa- ratus with a dart sac plus an accessory sac and mucus glands (C. frutis circassica (Mousson, 1863)), only mucus glands (C. frutis frutis) or none of these accessory genital organs (C. frutis veselyi (Frankenberger, 1919)). Schileyko (1978) classified these morphotypes as subspecies, whereas Giusti and Manganelli (1987) questioned whether they are related at all. Hausdorf (2001) followed Schileyko (1978), but questioned whether the morphotypes are actually genetic entities, because their ranges interdigitate in western Georgia. Furthermore, Hausdorf (2001) separated C. lasistana from C. frutis because of the much shorter spermatophore-forming flagellum. http://dx.doi.org/10.1016/j.ympev.2015.07.012 1055-7903/Ó 2015 Elsevier Inc. All rights reserved. q This paper was edited by the Associate Editor Jan Strugnell. ⇑ Corresponding author. E-mail address: hausdorf@zoologie.uni-hamburg.de (B. Hausdorf). Molecular Phylogenetics and Evolution 93 (2015) 129–142 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev