Journal of Molluscan Studies Advance Access published 4 December 2013 Journal of The Malacological Society of London Molluscan Studies Journal of Molluscan Studies (2013) 1–11. doi:10.1093/mollus/eyt039 A CONTRIBUTION TO THE PHYLOGENY OF ALBANIAN AGATHYLLA (GASTROPODA, CLAUSILIIDAE): INSIGHTS USING MORPHOLOGICAL DATA AND THREE MITOCHONDRIAL MARKERS ZOLTA´ NFEHE´ R1, ARISTEIDIS PARMAKELIS2, MARIA KOUTALIANOU2, 2 3 1 THANOS MOURIKIS ,ZOLTA´ NPE´ TER ERO´ ´ SS AND VIRA´ G KRI´ ZSIK Downloaded from 1Hungarian Natural History Museum, 13 Baross utca, H-1088 Budapest, Hungary; 2Department of Ecology and Taxonomy, Faculty of Biology, University of Athens, Panepistimioupoli Zografou, GR-15784 Athens, Greece; and 336 Bem utca, H-1151 Budapest, Hungary Correspondence: Z. Fehe´r; email: [email protected] http://mollus.oxfordjournals.org/ (Received 16 May 2013; accepted 28 August 2013) ABSTRACT In the present study, we revised Albanian Agathylla, combining the traditional shell morphological ap- proach with a phylogenetic reconstruction based on three mitochondrial DNA markers (COI, COII and 16S rRNA). From the faunistic perspective, Albania was an almost completely unexplored country until the end of the 1990s. Following recent intense field sampling activity in Albania, the number of known Agathylla populations has increased, providing an opportunity to investigate how additional ma- terial can contribute to the resolution of the taxonomy of the Albanian Agathylla species. Newly discov- by guest on December 7, 2013 ered populations indicate greater morphological variability than previously recorded and highlight the presence of various other character state combinations. Based on these findings, we argue that only two Agathylla taxa occur in the region, namely A. neutra and A. biloba. Due to apparent morphological differ- ences, they are still treated as distinct species, but high intraspecific morphological variability makes it unjustifiable to delimit subspecies. The molecular phylogeny indicates a close relationship between A. neutra and A. biloba, but could neither confirm nor refute their reciprocal monophyly, so their possible conspecificity requires further evaluation. INTRODUCTION group. This is of special importance in the case of groups that are difficult to locate (e.g. because of cryptic behaviour, or inaccess- Taxon delimitation is a crucial issue in biodiversity research ible habitat) or distributed in less explored regions (Criscione & because accurate taxon boundaries are of fundamental import- Ko¨ hler, 2013). ance in several fields of supraindividual biology. At present, Rock-dwelling door snails (Clausiliidae), including Agathylla advanced molecular-based approaches (Hebert et al., 2003; Pons species, occurring in Albania are such a case for several reasons. et al., 2006; Craft et al., 2010; Esselstyn et al., 2012; Puillandre Firstly, their systematics is exclusively based on a typological ap- et al., 2012a, b), especially within the framework of integrative proach. Secondly, they are predominantly found on isolated taxonomy, are gaining ground. Integrative taxonomy (the de- limestone outcrops, a habitat type that is spatially scattered limitation of species in a single framework combining different across wide areas of relatively inaccessible terrain. Thirdly, facets of species identity including morphology, ecology, bio- because from a faunistic perspective Albania is the most unex- geography and genetic data; Padial et al., 2010; Puillandre et al., plored region in Europe, since the country was not really access- 2012a, b), provides a comprehensive toolkit to define taxon ible until the 1990s. boundaries. Whereas some clausiliid genera occurring in the Balkans have However, for a long time taxonomy was based almost exclu- been used in numerous biogeographical, ecological and evolu- sively on morphological characters and it is still a fact that the tionary studies (Schilthuizen & Lombaerts, 1994; Schilthuizen vast majority of currently acknowledged taxa have only been & Lombaerts, 1995; Schilthuizen et al., 2004; Uit De Weerd & described in the traditional way. In addition, relatively little Gittenberger, 2004; Uit De Weerd, Piel & Gittenberger, 2004; attention has been given to the question of how these taxon Uit De Weerd & Gittenberger, 2005; Uit De Weerd, Schneider delimitations are affected by the intensity of study of a certain & Gittenberger, 2005; Uit De Weerd et al., 2006; Douris et al., # The Author 2013. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved Z. FEHE´ R ET AL. 2007; Pa´ ll-Gergely, Kornilios & Giokas, 2012; Fehe´ r et al., Agathylla species (14 of the 24 known taxa) were included in the 2013), Agathylla Adams & Adams, 1855 remains poorly studied. molecular analysis to infer the relationship of the two focal Uit de Weerd & Gittenberger (2013) have recently found that species as well as their position within the whole genus. Agathylla exhibits affinities with Apennine genera like Muticaria, Leucostigma and Lampedusa, but little is known about phylogenet- MATERIAL AND METHODS ic relationships within the genus. Agathylla is distributed along the western Adriatic coast from Origin of specimens Dalmatia (Croatia) to Epirus (northwestern Greece). Available The majority of the examined material was collected recently and distribution data are very scarce, most of the Agathylla taxa being is deposited in the Hungarian Natural History Museum (HNHM). known from only a few locations (Wagner, 1922; Nordsieck, In case of taxa with well-defined type localities, we attempted 1972; Zilch, 1977; Karaman, 2006). Until recently, this was also to sample topotypical populations as well. Additionally, we the case with the two species A. biloba and A. neutra that are dis- have investigated the material of the collections of the NHMW. tributed mainly in Albania. Sampling localities of recently collected material are listed as pre- Agathylla neutra, a small and smooth-shelled clausiliid species, cisely as possible; geographic coordinates, if available, are given in was first described by Westerlund (1898) as Clausilia (Delima) the format in which they were recorded (Systematic descriptions neutra, from the Tzoumerka mountains (northern Greece). That and Table 1). Information from the labels of old museum speci- population has not been collected again for a long time and, due mens is given in double quotation marks and, if necessary, expla- to the short and uninformative description and lack of available nations are provided within square brackets. For brevity, names type material in well-known collections, this species has long been of the frequently encountered collectors are abbreviated as follows: forgotten. The name was reintroduced more than a century later Z.E. ¼ Zolta´ nPe´ ter Ero´ ´ss, Z.F. ¼ Zolta´ nFehe´ r, A.H. ¼ Andra´ s by Nordsieck (2001) following the discovery of some topotypical Hunyadi, T.H. ¼ Tama´ sHusza´ r, J.K. ¼ Jeno´ ´ Kontscha´ n, Downloaded from material in the collections of the Naturmuseum Senckenberg in K.K. ¼ Korne´ lKova´ cs, D.M. ¼ Da´ vid Mura´ nyi. Frankfurt am Main and the Naturhistorisches Museum Wien (NHMW). In the meantime, other authors, being unaware of Morphological descriptions A. neutra, described some related taxa. In 1914, Wagner described two taxa from northern Albania, a ribbed one as Alopia (Agathylla) We compared the newly collected material with the type lots and/or the topotypic material as well as with the descriptions in biloba and a smooth-shelled one as its subspecies, Alopia http://mollus.oxfordjournals.org/ (Agathylla) biloba merditana (Sturany & Wagner, 1915). In 1996, the literature (Westerlund, 1898; Sturany & Wagner, 1915; Brandt, 1961; Nordsieck, 1996; Gittenberger, 1998; Nordsieck, a smooth-shelled population was found in C¸orovode¨ (southern Albania), resulting in the elevation of A. merditana to species level 2001). We gave special attention to morphological features such as shell size, width/height (W/H) ratio, sculpture of the upper and to the description of the C¸orovode¨ population as its subspe- cies, A. merditana scraparana (Nordsieck, 1996). The same taxon whorls and the cervical part, palatal callus, cervical swelling, was reported to occur in two further locations (Leskovik and subsutural swelling, structures of the lunella, the upper palatal Loutra) near the Albanian–Greek border (Gittenberger, 1998). fold and the basal fold, shape and protrusion of the aperture, When Nordsieck (2001) reintroduced A. neutra, it was considered that are considered important taxonomic features of the group. as the senior synonym of A. merditana scraparana. DNA extraction, amplification and sequence determination As regards A. biloba, Wagner’s syntype material originated from two localities, “Skutari” [¼Shkode¨ r] and “Vaudenjs am Nine Agathylla species (29 populations) and two outgroup by guest on December 7, 2013 Drin bei Skutari” [¼Vau i Deje¨ s]. Brandt (1961) subsequently species (Siciliaria pantocratoris margaritifera and Herilla illyrica ori- described a subspecies, A. biloba dabovici, from “Fluß Shen”. Later bates) were included in the molecular analyses. The specimens on, an A. biloba population found in the Fangul Pass (central were preserved either in absolute or 70% ethanol. Total Albania) was described as A. biloba martae (Ero´ ´ss, Fehe´ r& genomic DNA was isolated from the foot muscle of one adult in- Szekeres, 1999). The most recent systematic concept of this group dividual from each population (Table