Revision of the Helicellinae of Crete (Gastropoda: Hygromiidae) BERNHARD HAUSDORF* and JAN SAUER Zoological Museum, University of Hamburg, Martin-Luther-King-Platz 3, D-20146 Hamburg, Germany Received 17 April 2008 accepted for publication 31 July 2008 This paper presents a comprehensive revision of the Helicellinae of Crete (Gastropoda: Hygromiidae) based on characters of the shell and the genitalia. The native fauna includes eleven Xerocrassa species, two Pseudoxerophila species, and perhaps one Xeromunda species. One additional Xerocrassa species and eight species of the genera Trochoidea, Xeropicta, Xerolenta, Xerotricha, Microxeromagna, and Cernuella were probably introduced by man to Crete. The distribution patterns of the endemic Xerocrassa and Pseudoxerophila species do not provide evidence for the hypothesis that these radiations were caused by the fragmentation of the region of present-day Crete into several palaeoislands in the late Miocene and Pliocene. �� 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 157, 373���419. doi: 10.1111/j.1096-3642.2008.00504.x ADDITIONAL KEYWORDS: biogeography ��� Greece ��� Pseudoxerophila ��� radiation ��� taxonomy ��� palaeogeo- graphy ��� Xerocrassa. INTRODUCTION The land snail fauna of Crete is extraordinarily rich. Compared with the land snail fauna of other Aegean islands, the species number on Crete is much higher than would be expected from its area (Welter-Schultes & Williams, 1999). The high species richness of land snails on Crete is mainly the result of a few radia- tions. The radiations of the genera Albinaria Vest, 1867 (Clausiliidae Gittenberger, 1991 Schilthuizen & Gittenberger, 1996 Douris et al., 1998 Welter- Schultes, 2000a Nordsieck, 2004 Schilthuizen et al., 2004), Mastus Beck, 1837 (Enidae Maassen, 1995 Welter-Schultes, 2000b Parmakelis et al., 2005), and Orculella Steenberg, 1925 (Orculidae Gittenberger & Hausdorf, 2004) on Crete have already been studied in detail. Maltzan (1883, 1887) identified at least one radiation of xerophilous hygromiids on Crete. However, after Maltzan, this radiation has been almost completely neglected. It is still unknown how many xerophilous hygromiid species live on Crete, how they can be delimited, and where they are distributed. Because the anatomy of most of the described species is unknown, it is even unclear to which genera these species belong. As a first step towards a better understanding of the evolution of the Cretan xerophilous hygromiids, we present a compre- hensive revision of the Helicellinae s.s. of Crete. The Helicellinae Ihering, 1909 (Gastropoda: Hygro- miidae) differ from most other Hygromiidae in the course of the right ommatophoral retractor, which does not cross between the penis and the vagina, and in the whitish shell. The systematics of the Helicelli- nae traces back to Hesse (1926, 1934), who included three genera in the subfamily, Helicella F��russac, 1821, Cochlicella F��russac, 1821, and Monilearia Mousson, 1872, which is closely related to Cochlicella. Hesse divided Helicella s.l. into several subgenera that are mainly based on differences in the genitalia. Later, Helicella s.l. was further divided into several genera that were mainly based on the number of dart and accessory sacs (e.g. Zilch, 1960). Some authors (e.g. Zilch, 1960) also included Monacha Fitzinger, 1833 in the Helicellinae, because its right ommato- phoral retractor does not usually cross between the *Corresponding author. E-mail: hausdorf@zoologie.uni-hamburg.de Zoological Journal of the Linnean Society, 2009, 157, 373���419. With 15 figures �� 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 157, 373���419 373

penis and the vagina (but see Hausdorf, 2000 for exceptions). Schileyko (1972, 1978, 1991) transferred Monacha, and other genera of the Hygromiidae in which the dart apparatus is transformed to appen- diculae (but in which the right ommatophoral retrac- tor crosses between the penis and the vagina), into a separate subfamily of the Hygromiidae. Because of the structure of the stimulator, he excluded Cochli- cella from the Hygromiidae, and established a separate family for Cochlicella and Monilearia. Fur- thermore, he supposed that the course of the right ommatophoral retractor and the whitish shell are adaptations to dry habitats. He concluded that even the remaining Helicellinae s.s. (i.e. without Cochli- cella, Monilearia, and Monacha) are polyphyletic, and that the xerophilous groups with a free right ommato- phoral retractor and a whitish shell originated convergently from various more hygrophilous hygromiids, which resemble the individual xerophil- ous groups in the structure of the dart apparatus. This view has essentially been accepted by Nordsieck (1987, 1993), who, however, included the Cochlicella group in the Monachainae Wenz, 1930 (1904). In contrast, Hausdorf (1988) considered the Helicellinae s.s. to be monophyletic, because they differ from the Hygromiinae Tryon, 1866, Monachainae, and Cochli- cellinae Schileyko, 1972 in chromosome numbers (n = 26 or 27, rarely 24 or 25, in the Helicellinae n = 23, rarely 21, 24, or 26, in the Hygromiinae, Monachainae, and Cochlicellinae). A phylogenetic analysis of partial cox1, 16S rDNA, 18S rDNA, and internal transcribed spacer 1 (ITS1) sequences revealed the monophyly of Helicellinae, inclusive of Monacha (Steinke, Albrecht & Pfenninger, 2004). However, the bootstrap value for that clade is below 50%. Phylogenetic analyses of partial 16S rDNA sequences alone were insufficient to decide whether or not the Helicellinae are monophyletic (Manganelli, Salomone & Giusti, 2005). However, this study sug- gested that Monacha is more closely related to Brady- baenidae than to Helicellinae. A phylogenetic analysis of partial 28S rDNA sequences (Koene & Schulen- burg, 2005) confirmed the monophyly of Helicellinae, and showed that Monacha is more closely related to some Hygromiinae than to Helicellinae. Accordingly, we consider Helicellinae s.s., i.e. without Cochlicella and Monacha, in this paper. MATERIAL AND METHODS The material on which this revision is based was collected on three expeditions to Crete in July/August and September/October 2004, and September/October 2005. During these expeditions, land snails were sampled at about 500 localities across Crete. Among others, about 1250 xerophilous hygromiid lots were collected, and are now kept in the Zoological Museum of the University of Hamburg (ZMH). Living speci- mens were killed by putting the crawling animal into boiling water the soft parts were then conserved in 70% ethanol. Additional 3650 lots (mainly shells) were borrowed from the following collections: Haus der Natur, Cismar (HNC) Hungarian Natural History Museum, Budapest [HNHM material determined as Xero- crassa cretica, Xeromunda candiota, and Cernuella virgata has only partly been revised] collection W. J. M. Maassen, Duivendrecht (MAA) Natur- historiska Museet, G��teborg (NMG) collection C. A. Westerlund in the NMG (NMGW) Nationaal Nat- uurhistorisch Museum, Leiden, formerly Rijksmu- seum van Natuurlijke Historie (RMNH) collection P. Subai, Aachen (SUB) Museum f��r Naturkunde, Berlin (ZMB). The material studied is listed in Appen- dix S1. The synonymy lists include only the original descriptions, and quotations concerning material from Crete or relevant for the interpretation of the species. Photographs were taken with the digital camera Leica DFC320 mounted on a binocular microscope (Leica MZ16) using the software IM50 v4.0 (Leica Microsystems Imaging Solutions). Shell measure- ments were taken from digital photographs using the program analySIS Pro v3.2 (Olympus Soft Imaging Solutions) or with an ocular micrometer. The counting of the shell whorls (exactness 1/4 whorl) follows the method of Kerney, Cameron & Jungbluth (1983). Mor- phometric data were analysed with SPSS v15.0 (SPSS Inc.). The terminology used to describe the genitalia is illustrated in Figure 1. The terms proximal and distal refer to the position in relation to the gonad. The measurements of the individual parts of the genitalia were taken with an ocular micrometer. The measure- ments were usually repeated once. The lengths of the various parts of the genitalia correlate with body size, and, therefore, are often not species specific. However, the proportions of the distal parts of the genitalia proved to be important species-specific characters. In all genera except Xerocrassa and Trochoidea, the penis and the distal epiphallus up to the insertion of the penial retractor have been measured together, because the penis���epiphallus boundary cannot be rec- ognized in these groups without an examination of the inner structure. The genitalia were not described for each species in detail, because there are usually only morphometric differences between closely related species. However, all measurements are listed to facilitate future taxo- nomic work. 374 B. HAUSDORF and J. SAUER �� 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 157, 373���419

Most measurements of the genitalia were taken from material collected by us and fixed in the same way (see above). However, we did not note any sys- tematic deviations of the morphometric characters that we consider to be species specific (especially the ratios of different parts of the genitalia) in specimens from other collections that were fixed in different ways (e.g. by drowning). The spelling of the geographic names is usually taken from the ���Kreta��� map, 1 : 140 000 (Reise Know- How Verlag, Bielefeld, 2002). The localities are arranged according to Nomos (N. prefectures) in the locality lists. Within Nomos, localities are ordered alphanumerically according to the 10-km Universal Transverse Mercator (UTM) code. We tested the hypothesis that the distribution areas of the recent endemic species are centred on Neogene palaeoislands by means of a Monte Carlo procedure. We used the null model proposed by Hausdorf & Hennig (2003), but modified it so that the species richness distribution of the geographic cells is not considered. This null model generates range data sets such that their range size distribution and the spatial autocorrelation of the occurrences of a species approach the parameters in the real data set. We used the number of occurrences of species in 10-km UTM grids that are located on areas formerly belong- ing to palaeoislands as the test statistic. If the recent species originated on Neogene palaeoislands, and if their ranges have not shifted, the number of occur- rences of species that are located on areas formerly belonging to palaeoislands should be higher in the real data set than in the simulated data sets, in which the ranges are placed independently of the position of the palaeoislands. The position of the palaeoislands was taken from the map of Crete in the Pliocene from Fassoulas (2001: fig. 11). SYSTEMATICS HELICELLINAE IHERING 1909 XEROCRASSA MONTEROSATO, 1892 Xerocrassa Monterosato, 1892: 23. Type species (by monotypy): Helix seetzeni L. Pfeiffer, 1847. Diagnosis: Xerocrassa is characterized by a symmetri- cal dart apparatus, consisting of two small accessory sacs and usually four branched glandulae mucosae around the vagina, irregular longitudinal folds at the inner side of the wall of the vagina, and the lack of a well-developed appendix at the atrium. The penis is innervated from the right cerebral ganglion. Remarks: The homology of the two small sacs at the vagina is doubtful. In a few Xerocrassa s.l. (as used by Nordsieck, 1993) species, e.g. Xerocrassa geyeri (So��s, 1926), there are small swellings at the base of these sacs in a position that is similar to the posi- tion of the dart sacs of Xeropicta. If these swellings were rudimentary dart sacs, the sacs of Xerocrassa and Trochoidea would be homologous to the acces- sory sacs of other Hygromiidae, and not to the dart sacs. Xerocrassa differs from Trochoidea in the lack of a well-developed appendix at the atrium. However, there are structures in the atrium of several Xerocrassa species that are probably homolo- gous with the inner structures of the atrial appen- dix of Trochoidea, and in some Xerocrassa species (e.g. X. cretica, X. franciscoi, X. heraclea) they even form a lateral bulge at the atrium. This is also the case in Xerocrassa siphnica (Kobelt, 1883), which has therefore been erroneously included in Tro- choidea s.s. by Fuchs & K��ufel (1936) (see also Mylonas et al., 1995). Xerocrassa in the wide sense, as used by Nordsieck (1993), is probably paraphyl- Figure 1. Terminology of the distal parts of the genitalia: ap, appendix at, atrium bc, bursa of the bursa copulatrix da, dart apparatus ep, epiphallus fl, flagellum fod, free oviduct gm, glandulae mucosae p, penis ped, pedunculus pr, penial retractor sod, spermoviduct vag, vagina vd, vas deferens. HELICELLINAE OF CRETE 375 �� 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 157, 373���419

etic with regard to Trochoidea, and needs a revision. Based on mitochondrial DNA sequences (J. Sauer & B. Hausdorf, unpubl. data), the Xerocrassa species from Crete belong to the same clade as the type species of Xerocrassa, Xerocrassa seetzeni (L. Pfe- iffer, 1847), from the Levant. Thus, the Cretan species will remain in Xerocrassa even after revi- sion. XEROCRASSA AMPHICONUS (MALTZAN, 1883) (FIGS 2A, 3A, 4A, 5A TABLES 1���3) Helix (Jacosta) amphiconus Maltzan, 1883: 102. Locus typicus: ���prope Sitia insulae Cretae���, Greece. Helix (Jacosta) amphiconus ��� Kobelt, 1888: 25, pl. 98, fig. 546. Helix (Xerophila) amphiconus ��� Martens, 1889: 187. Helicella (Trochoidea) syrensis amphiconus ��� Fuchs & K��ufel, 1936: 632. Helix (Jacosta) amphiconus ��� Lindner, 1994: 78, fig. 2. Trochoidea mesostena ��� Vardinoyannis, 1994: 85, 88, 130, map 45 (partim, non Westerlund, 1879). Type material: Syntypes: Greece, Kr��ti, N. Lasithi Sitia ��� Moni Toplou, MU29 (ZMB 39695/4). Diagnosis: Xerocrassa amphiconus is characterized by a strongly depressed, perforated shell with a protruding keel, a relatively short flagellum (proximal epiphallus : flagellum = 3.3���4.6), and a penial papilla with a long, terminally open basal part and a very short conical apical part, with an open channel. Shell (Fig. 2A Table 1): Strongly depressed conical with 41/4/43/4 whorls that are almost plain or even concave at the top side teleoconch with regular fine ribs and irregular impressions (especially under- neath) whitish, with or without brown bands that might be fused or break up into spots all whorls with a protruding keel aperture rhombic upper insertion of the peristome not or slightly descending peristome sharp, not expanded, with a whitish internal rib umbilicus prick-like, not obscured by the columellar edge. Genitalia (Figs 3A, 4A Tables 2 and 3): The penial papilla is divided into a long, slightly tapering, ter- minally open basal part and a very short conical apical part, with an open channel that is adnate with the basal part at the side opposite the opening. There is a glandular belt in the penial wall, near its proxi- mal end, and a longitudinal glandular field at the abvaginal inner side of the penis wall, with distinct folds. There are no distinct stimulatory structures in the atrium. KEY TO THE GENERA OF THE HELICELLINAE PRESENT ON CRETE 1a. With one dart apparatus...................................................................................................................2 1b. With two symmetrical dart apparatuses...............................................................................................4 2a. Accessory sac much smaller than dart sac, transformed into a cavity between the vagina wall, the dart sac, and a tissue-layer that envelopes parts of the vagina and the dart sac..............................................Xeromunda 2b. Accessory sac only slightly smaller than dart sac ..................................................................................3 3a. Dart apparatus with large, conical papilla inside the penis is innervated from the right pedal ganglion shell without hairs.......................................................................................................................Cernuella 3b. Dart apparatus without large papilla inside the penis is innervated from the right cerebral ganglion shell with hairs.........................................................................................................................Microxeromagna 4a. Dart apparatus with dart-bearing dart sac...........................................................................................5 4b. Dart apparatus without dart-bearing dart sac.......................................................................................7 5a. Dart sac much larger than accessory sac shell with hairs..........................................................Xerotricha 5b. Dart sac small, not much larger than accessory sac...............................................................................6 6a. Accessory sacs much longer than wide, sticking out from the vagina penis with appendix ................ Xeropicta 6b. Accessory sacs not much longer than wide, attached to a dilatation of the vagina penis without appendix....... ................................................................................................................................ Pseudoxerophila 7a. With large appendix at the atrium, two strong longitudinal folds surround each opening of the accessory sacs into the vagina at the inner side of the wall of the vagina, and fuse pairwise at their distal and proximal ends ................................................................................................................................. Trochoidea 7b. Without appendix, or at most with a small swelling at the atrium, inner side of the wall of the vagina with irregular longitudinal folds ................................................................................................... Xerocrassa 376 B. HAUSDORF and J. SAUER �� 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 157, 373���419

Remarks: Xerocrassa amphiconus and X. siderensis populate the same region in eastern Crete, but are usually not syntopic. Xerocrassa amphiconus is mainly distributed in the higher central region, whereas X. siderensis occurs primarily in the coastal region. However, there are exceptions to this pattern, e.g. occurrences of X. amphiconus near Agia Fotia and Kato Zakros, and records of X. siderensis near Ziros and Zakros. Only at Stavromenos, 2.1 km towards Katsidoni, a single X. amphiconus was found together with two X. siderensis. Apart from differing altitudi- nal preferences, we could not ascertain ecological differences between the two species. In contrast to the situation in X. siphnica, where keeled forms are said to aestivate only on or under stones, whereas rounded forms also aestivate under shrubs (Mylonas et al., 1995), both X. amphiconus and X. siderensis aestivate under stones as well as in shrubs. Xerocrassa amphi- conus and X. siderensis are very closely related. They do not form separate clades in trees based on mito- chondrial genes or amplified fragment length poly- morphism (AFLP) markers (J. Sauer & B. Hausdorf, unpubl. data). This means either that they have not been separated long enough for lineage sorting to be completed or that there is still introgression. In con- trast to other closely related Xerocrassa species from Crete, we have not found differences in the genitalia of X. amphiconus and X. siderensis. The differences in shell characteristics are no larger than differences between some extreme forms, classified as X. mesos- tena. However, in some areas, e.g. north of Ziros or south of Langada, populations of both taxa occur in close vicinity without continuous morphological tran- sitions. We have measured the large diameter of the shell, the small diameter of the shell (perpendicular to the large diameter), the diameter at three whorls, the width of the spire, the height of the shell, the height of the spire, the diameter of the aperture, the height of the aperture, the width of the umbilicus, and the number of whorls of twelve specimens from KEY TO THE XEROCRASSA SPECIES PRESENT ON CRETE 1a. Proximal epiphallus : flagellum 2.3.................................................................................................2 1b. Proximal epiphallus : flagellum 2.2.................................................................................................6 2a. Large shell diameter 9 mm proximal epiphallus : flagellum 3.0................Xerocrassa kydonia sp. nov. 2b. Different to above...........................................................................................................................3 3a. Shell with a protruding keel ............................................................................................................4 3b. Shell without a protruding keel........................................................................................................5 4a. Proximal epiphallus : flagellum ratio = 2.3���2.6 umbilicus 1.7���2.6 mm............Xerocrassa franciscoi sp. nov. 4b. Proximal epiphallus : flagellum = 3.3���4.6 umbilicus 0.2���1.0 mm..........Xerocrassa amphiconus (Maltzan, 1883) 5a. Penial papilla with a long, terminally open basal part and a very short conical apical part (Fig. 4A).............. ..................................................................................................Xerocrassa siderensis (Maltzan, 1883) 5b. Penial papilla with a broader basal part and a narrower apical part, with a terminal opening (Fig. 4E)......... ................Xerocrassa mesostena (Westerlund, 1879) (a few specimens from the surroundings of Ano Viannos) 6a. Large shell diameter, usually 13 mm if smaller, columellar edge of aperture almost perpendicular to body whorl, umbilicus almost concentric ..................................................... Xerocrassa cretica (L. Pfeiffer, 1841) 6b. Large shell diameter, 13 mm, with columellar edge of aperture obliquely converging to body whorl............7 7a. Umbilicus very narrow, partly or completely obscured by the columellar edge, whorls rapidly increasing, penial papilla cylindrical with a terminal opening..................................................Xerocrassa meda (Porro, 1840) 7b. Umbilicus narrow or moderately wide, whorls increasing more slowly .....................................................8 8a. Umbilicus almost concentric or only slightly eccentric, penial papilla with a cylindrical basal part and a dilated apical part, with a subterminal opening (Fig. 4C).................................................................................9 8b. Umbilicus narrow and/or eccentric, distinctly enlarged by the last whorl................................................10 9a. Shell striated or finely ribbed, without distinct keel large shell diameter : umbilicus width 0.15 proximal epiphallus : flagellum 1.2......................................................... Xerocrassa subvariegata (Maltzan, 1883) 9b. Shell coarsely ribbed, with a distinct keel large shell diameter : umbilicus width 0.14 proximal epiphallus : flagellum 1.1 .................................................................................. Xerocrassa grabusana sp. nov. 10a. Penial papilla with a long, terminally open basal part and a very short conical apical part, large shell diameter 8.5 mm (Fig. 4D).........................................................................................................................11 10b. Penial papilla with a broader basal part and a narrower apical part, with a terminal opening, large shell diameter usually 8.5 mm (Fig. 4E).............................................Xerocrassa mesostena (Westerlund, 1879) 11a. Total vagina length : vagina up to the base of the dart apparatus 1.6..........Xerocrassa heraklea sp. nov. 11b. Total vagina length : vagina up to the base of the dart apparatus 1.5.................................................12 12a. Proximal epiphallus : total vagina length 2.1........................................ Xerocrassa rhithymna sp. nov. 12b. Proximal epiphallus : total vagina length 2.3.......................................Xerocrassa lasithiensis sp. nov. HELICELLINAE OF CRETE 377 �� 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 157, 373���419