Europe PMC Funders Group Author Manuscript J Molluscan Stud. Author manuscript; available in PMC 2014 September 04. Published in final edited form as: J Molluscan Stud. 2011 February 1; 77(1): 30–40. doi:10.1093/mollus/eyq037. E GENETIC DIFFERENTIATION AND SHELL MORPHOLOGY OF u r o p TROCHULUS OREINOS (WAGNER, 1915) AND T. HISPIDUS e P (LINNAEUS, 1758) (PULMONATA: HYGROMIIDAE) IN THE M C F NORTHEASTERN ALPS u n d e MICHAEL DUDA1, HELMUT SATTMANN1, ELISABETH HARING1,2, DANIELA BARTEL1,2, r s A HANS WINKLER3, JOSEF HARL1, and LUISE KRUCKENHAUSER1 u t 1Museum of Natural History Vienna, Burgring 7, A-1010 Vienna, Austria h o r M 2Department of Evolutionary Biology, University of Vienna, Althanstr. 14, A-1090 Vienna, Austria a n 3Konrad Lorenz Institute for Comparative Ethology, Austrian Academy of Sciences, u s c Savoyenstraße 1A, A-1160 Vienna, Austria r i p t s Abstract Trochulus oreinos oreinos and T. oreinos scheerpeltzi are two land snail taxa endemic to the Northeastern Austrian Alps, which have been regarded as subspecies of the highly variable, widespread land snail T. hispidus. We analysed these three taxa morphologically and genetically to evaluate whether a delimitation between them is possible and, if so, to resolve their phylogenetic relationships. Shell morphological results revealed high similarity between the two E T. oreinos taxa, and that they are clearly separated from T. hispidus. Additionally, the T. oreinos u ro subspecies concur with respect to their habitat preferences, as they are both restricted to rocky p e high alpine areas, whereas the local form of T. hispidus is distributed over a wider altitudinal P M range in moist areas and scrubby perennial herb vegetation near water bodies. While the C morphological and ecological results allow clear differentiation between T. hispidus and T. F u oreinos only, analyses of the mitochondrial cytochrome c oxidase subunit I and 16S rRNA genes n d revealed high sequence divergences between all three taxa, which indicates that they represent old e r s lineages. The two T. oreinos taxa appear as distantly related sister groups, well separated from T. A u hispidus. Whether T. o. oreinos and T. o. scheerpeltzi should be considered as species cannot be t h decided at the current state of knowledge. o r M a n u INTRODUCTION s c r i A large number of land snail species have been described from the Alpine region, including p t s several endemics. Many species have been divided into different infra- or subspecific entities (races, forms etc.), mainly by minor shell morphological features. This © The Author 2010. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Correspondence: M. Duda, [email protected]. DUDA et al. Page 2 differentiation has often been explained by glacial/postglacial events of isolation, displacement, dispersal and (re-)colonization (Adensamer, 1937; Klemm, 1974; Gittenberger, 1991). Moreover, the environment may have triggered special genetic adaptations and/or changes caused by phenotypic plasticity, e.g. smaller shells at higher elevations. Often, subspecific entities have been described from defined altitudinal zones or E within the boundaries of particular mountain massifs. Different interpretations and opinions u concerning the origin of described morphological variants often resulted in controversial r o p taxonomic conclusions and systematic assignments. e P M The genus Trochulus Chemnitz, 1786 is one such example. The genus comprises small, C F pulmonate land snails with flattened or globular, particularly hairy shells, mainly distributed u in Central and Western Europe. The CLECOM list (Falkner, Bank & von Proschwitz, 2000) n d e gives up to 18 different species of Trochulus s. s. for central and northern Europe. Two r s A species – Trochulus waldemari (Wagner, 1915) and T. suberectus (Clessin, 1878) – were not u included by Falkner et al. (2000), because their distribution ranges are not covered by the t h o CLECOM list. However, the number of recognized species varies among different authors. r M For example, one of the listed taxa, T. alpicola (Eder, 1921), has not been regarded as an a n independent species by some authors. Additionally, some authors (e.g. Davis, 2004; u s Cameron et al., 2006) have expressed objections to some taxonomic decisions in the c r i CLECOM list. Subsequently, Proćków (2009) enumerated 22 species in Trochulus by p t s lumping T. plebeius and T. hispidus at species level and the (sub)genera Petasina and Plicuteria at generic level, based on morphological data. This study is focused on the poorly known northeastern Alpine endemic T. oreinos (Wagner, 1915), which was originally regarded as regional subspecies of T. hispidus (Linnaeus, 1758), but was later considered as a separate species not closely related to T. hispidus (Falkner, 1982, 1995). According to the current taxonomic view, T. oreinos comprises two subspecies, E u T. oreinos oreinos (=Fruticicola hispida oreinos) and T. oreinos scheerpeltzi (Mikula, 1957) r o (=T. hispidus scheerpelzi), which are both endemic to the northeastern Alps in Austria (Fig. p e P 1A, B). Falkner (1982, 1995) based his decision to treat T. o. scheerpeltzi as a subspecies of M T. oreinos on ‘differing hair morphology’ of the two T. oreinos taxa. However, he did not C F provide any details. This is remarkable because the original description of Wagner (1915) u characterized T. o. oreinos as hairless, and Mikula (1957) did not mention any hairs in T. o. n d e scheerpeltzi. Because no clear descriptions and pictures are available, knowledge of T. o. r s A oreinos and T. o. scheerpeltzi has been restricted to a few specialists who have inspected u specimens collected by the describers and deposited in scientific collections. t h o r M Trochulus o. oreinos (Fig. 1A) is found in Lower Austria and Styria at high altitudes (1,600– a 2,280 m). Its distribution extends from Schneeberg mountain to Totes Gebirge (Klemm, n u 1974). Reischütz & Reischütz (2009) mentioned rocky grass biotopes and duff as habitats. s c r In the original description Wagner (1915) characterized it by a shiny, finely granular, i p ts hairless shell with coarse, irregular ridges. The shell was described as smaller than that of T. hispidus, but ‘more stable’. As additional traits Wagner mentioned a strong lip inside the aperture, visible as a yellow structure from outside, with a tooth-like structure at the basal margin. The type locality is at Hochschwab mountain in Styria at elevations above 2,000 m. So far, no picture of T. o. oreinos has been published. J Molluscan Stud. Author manuscript; available in PMC 2014 September 04. DUDA et al. Page 3 Trochulus o. scheerpeltzi (Fig. 1B) is found in the mountain ranges of Höllengebirge to Totes Gebirge and in parts of Haller Mauern (Klemm, 1974). Like T. o. oreinos this subspecies is found at high altitudes (1,600–2,300 m) and in a similar habitat – rocky grass biotopes and crevices with duff (Reischütz & Reischütz, 2009). In the original description Mikula (1957) mentioned a groove beneath a clearly visible keel as a trait distinguishing T. E o. scheerpeltzi from T. o. oreinos and T. hispidus. The type locality of T. o. scheerpeltzi is u ‘Hauptkar’ at the Hohe Nock Mountain in Upper Austria at elevations of 1,600–1,800 m. r o p The only published pictures are those in the original description (Mikula, 1957). e P M Trochulus hispidus (Fig. 1C) has a wide distribution in Europe, occurring over a broad range C F of altitudes (up to 2,300 m) and habitats. The distribution covers large parts of Europe from u Ireland and France to Kazan and St Petersburg in European Russia. In the north it reaches n d e the Arctic Circle. It does not occur in southernmost parts of Europe (Ložek, 1956). r s A According to Giusti & Manganelli (1987) records from Sardinia are very likely due to u confusion with Ichnusotricha berninii. As T. hispidus is a polymorphic species, its t h o systematics have long been the focus of controversy. Forcart (1965) suggested a division of r M Trichia hispida (nowadays Trochulus hispidus) into two distinct species, Trichia hispida and a n Trichia concinna. Subsequently, he assigned the two subspecies oreinos and scheerpeltzi to u s T. concinna. However, Gittenberger, Backhuys & Ripken (1970), followed by various c r ip authors including Klemm (1974), Falkner (1982) and Naggs (1985), rejected this theory t s because large clinal transition zones exist between hispida and concinna and the geographic distribution ranges are not clearly delimited. Shileyko (1978) raised doubts about the justification of the species status of several taxa of the ‘T. hispida group’ (including T. plebeia, T. sericea, T. septentrionalis and T. concinna). Recent papers that have employed molecular biological methods (Pfenninger et al., 2005; Dépraz, Hausser & Pfenninger, 2009) have provided an even more confusing picture, showing several distinct mitochondrial E lineages in T. hispidus, and perhaps the occurrence of cryptic species. Proćków (2009) u synonymized T. plebeius and T. concinnus with T. hispidus based on an extended r o p morphological analysis. e P M The questions we wanted to clarify were: (1) Can the three taxa be differentiated C F morphologically? (2) Are the two T. oreinos taxa and the northeastern Alpine form of T. u hispidus genetically differentiated? (3) Do the morphological and genetic data corroborate n d e the species status of T. oreinos? (4) Do the three forms occupy different habitats, elevations r s A and geographic ranges? Using samples covering the entire distribution range of T. o. oreinos u and T. o. scheerpeltzi as well as the local forms of T. hispidus we performed morphological t h o and genetic analyses to answer these questions. r M a The systematics of some species of Trochulus are problematic. This is especially true of T. n u hispidus with a number of divergent lineages (Pfenninger et al., 2005; Dépraz et al., 2009), s c r among which there may be some cryptic species. However, since no comprehensive i p ts phylogenetic study of Trochulus has yet been carried out, we provisionally adopted the classification of the Austrian taxa provided by Reischütz & Reischütz (2007). J Molluscan Stud. Author manuscript; available in PMC 2014 September 04. DUDA et al. Page 4 MATERIAL AND METHODS Study area and sampling The study area was located in the Northeastern Austrian Alps, including parts of the provinces Upper Austria, Lower Austria and Styria. This covers the distribution ranges of E the two T. oreinos taxa (according to Klemm, 1974) and adjacent areas where T. hispidus u occurs (Fig. 2). Most sample sites are situated on the limestone bedrock of the Northern r o p Calcareous Alps, one on Palaeozoic limestone of the Grazer Bergland and four on e P metamorphic rocks of the Central Alps. Most of the study area is characterized by a cool M humid Central European climate with heavy precipitation; only the easternmost and C F southeastern parts are influenced by the warmer and dryer Illyrian and Pannonian climates u n (Kilian, Müllner & Starlinger, 1994). Both T. oreinos subspecies occur down to an altitude d e of 1,300–1,450 m, which is also the lower limit of the subalpine zone. Like other marginal r s A Alpine areas, the study area has been the focus of research projects seeking potential glacial u t refugia (e.g. Schönswetter et al., 2005), because large parts of it remained ice-free during the h o r last glaciations (Van Husen, 1997). M a n Sampling and habitat analysis were carried out at 45 sampling sites (Fig. 2, Table 1). u s Topotypes of T. o. oreinos (11 specimens from sample site 32) and T. o. scheerpeltzi (11 c r i specimens from sample site 38) were included. Exact positions and elevations of collection p t s sites were determined using GPS. The sampling period extended from May to July in 2007, 2008 and 2009. Adjacent water bodies, vegetation, habitat structure and dominant plant species were recorded. Trochulus hispidus was identified by morphological traits described in the literature (e.g. Ložek, 1956; Gittenberger et al., 1970; Kerney, Cameron & Jungbluth, 1983). Trochulus o. oreinos and T. o. scheerpeltzi were identified using the original descriptions (Wagner, 1915; Mikula, 1957) as well as by comparison with reference E specimens (paratypes and syntypes) in the collections of the Natural History Museum, u Vienna (NHMW). In total, 327 specimens (225 living animals and 102 empty shells) of the r o p three Trochulus taxa were included (Table 1). e P M Shell morphology C F Four shell traits were measured in intact adult specimens (shell diameter, umbilicus u n diameter, shell height and height of last whorl) with a graduated eyepiece in a d e r stereomicroscope. The definition of adulthood from shell apertural traits was problematic, as s A presumably adult specimens of T. hispidus often lack an outer lip (Geyer, 1915; Frömming, u t 1954; Cameron, 1982). Therefore, individuals were defined as ‘adult’ when their shells had h o r a minimum diameter of 5.4 mm, as this was the size of the smallest individual seen with a M fully developed lip. This might appear arbitrary, because the standard literature and some a n u collections contain only ‘typical’ specimens with outer lip. However this analysis was s c intended to include all naturally occurring variants and therefore we had to define a size r i p limit. Altogether 304 specimens (complete specimens and empty shells) from all 45 sample t s sites were measured and standard variables (mean, variance and standard deviation) were calculated. Hairs (which are present also in juveniles) were inspected in all 327 individuals. Another two adult specimens could be investigated only genetically, because their shells were broken. A total of 111 specimens were investigated using all methods (Table 1). The J Molluscan Stud. Author manuscript; available in PMC 2014 September 04. DUDA et al. Page 5 measurement error is too often neglected in measurements of small (<10 mm) globular shells. The main source of error is the lack of precise measurement points on the shell. Furthermore, the definition of the main axes is not very precise and the projection of the shell in two dimensions is problematic. Measurement error was determined by repeated measurements (10 times) of shell diameter, umbilicus diameter and shell height in 140 E empty shells of Trochulus and examination of the distribution of residuals (total 4,200). u r o Four additional shell traits were recorded as presence/absence, because they were mentioned p e P as typical for T. o. oreinos in the original description (Wagner, 1915). Three of these were M apertural traits: basal tooth, internal rib and paler area around the aperture (Fig. 3). The C F occurrence of coarse, irregular ridges was also recorded as presence/absence. Ridges were u classified as ‘coarse’ if broad ridges (>0.5 mm) were immediately followed by smaller ones n d e (Fig. 3). In T. o. scheerpeltzi a groove beneath the keel was described by Mikula (1957) and r s A was recorded by us in three categories: well developed, partly developed and absent. If the u groove was clearly visible with ×16 magnification and covered at least 50% of the t h o circumference, it was characterized as well developed. If it covered less than this and was r M only weakly visible, it was characterized as partly developed. Measurements of shells were a n log-transformed. These data together with scores obtained with a correspondence analysis of u s presence/absence data were used in a canonical discriminant analysis (Tabachnick & Fidell, c r i 1996). Morphometric analyses were performed with programs written by one of us (H.W.). p t s Since we were interested in identifying group differences and the variables responsible for these differences, we used discriminant analysis rather than PCA or ordination techniques that deal with overall variation, which might be dominated by variables that do not contribute to, or even mask, the variation among groups. To quantify hair length and structure, digital microscopic images were taken of five hairs of 15 specimens (five of each form). Hair lengths were measured by using TPSdig Version E 2.14 (Rohlf, 2001). To proof the repeatability of measurements, all hairs were measured u r o twice. p e P M As measuring of hairs takes a lot of time and the different hair morphologies can be easily C recognized (Fig. 3), the values from all specimens were assigned to three categories: long F u hairs (>0.2 mm), short hairs (<0.1 mm) and no hairs. n d e r Genetic analysis s A u From 132 specimens (adults and juveniles) a partial region of the mitochondrial cytochrome t h c oxidase subunit I (COI) gene was sequenced. In addition, from representatives of each o r M clade a partial region of the mitochondrial 16S rRNA (16S) gene was sequenced (altogether a 38). As outgroup taxa Monacha cantiana and Plicuteria lubomirskii (one specimen each) n u s were analysed for both fragments. c r i p t A piece of foot tissue was extracted with QIAgen Blood and Tissue Kit. Primers were based s on those used by Gittenberger, Piel & Groenenberg (2004) for COI and by Pfenninger, Posada & Magnin, (2003) for 16S. Primers were optimized on the basis of alignments of published sequences of several snail species. Primer sequences for COI: COIfolmerfwd 5′- GGTCAACAATCATAAAGATATTGG-3′ (LCO1490 modified from Folmer et al., 1994) J Molluscan Stud. Author manuscript; available in PMC 2014 September 04. DUDA et al. Page 6 and COIschneckrev 5′-TATACTTCTGGATGACCAAAAAATCA-3′ (H2198-Alb modified from Gittenberger et al., 2004). Primer sequences for 16S: 16Sfw 5′- CGCAGTACTCTGACTGTGC-3′ (Pfenninger et al., 2003) and 16S_sch_rev 5′-CG CCGGTCTGAACTCAGATC-3′ (16Srev modified from Pfenninger et al., 2003). Resulting fragment sizes were 705 bp (COI) and about 395 bp (16S), respectively. PCR was E performed on a Master gradient thermocycler (Eppendorf) in 50 μl with 1 U Taq DNA u polymerase (Roche), 1 μM of each primer and 0.2 mM of each dNTP (Boehringer r o p Mannheim). Each PCR comprised 35 reaction cycles with the following annealing e P temperatures: 50°C (COI) and 55°C (16S). Control reactions for both DNA extractions and M PCR amplifications were carried out. PCR products were purified using the QIAquick PCR C F Purification kit (QIAGEN) and analysed with direct sequencing (both directions). u n Sequencing using the amplification primers was performed by AGOWA (Berlin, Germany). d e r s A For the COI sequences the alignment was straightforward as there were no insertions or u deletions. Alignment of 16S sequences was performed with Tcoffee (Notredame, Higgins & t h o Heringa, 2000) and adjusted manually. Neighbour-joining (NJ; Saitou and Nei, 1987) r M dendrograms were calculated with ClustalX v. 2.0.12 (Larkin et al., 2007) using p-distances. a n Bootstrap analyses were performed with 1,000 replicates. For calculation of models of u s sequence evolution the sequences were collapsed to haplotypes using Collapse1.2 (Posada, c r i 2004). The resulting dataset was used applying the Akaike information criterion corrected p t s for small sample size (AICc) as implemented in the jModelTest v. 0.1.1. (Posada, 2008); the selected models were HKY + I + G for the COI dataset, GTR + G for the 16S dataset and GTR + I + G for the combined (COI + 16S) dataset. Bayesian analyses (BI) were performed using MrBayes v. 3.1.2 (Huelsenbeck & Ronquist, 2001). Runs were started with random trees and performed for 2 million generations each with four Markov chains, and a sampling frequency of every 100th generation. Those trees generated prior to stationarity were discarded as burn-in and were not included in the calculation of the consensus trees. E u r o Numbers of haplotypes and haplotype diversity and average p-distances (gaps excluded) p e P were calculated with ARLEQUIN v. 3.11 (Excoffier, Laval & Schneider, 2005). The M sequences determined in this study are deposited in GenBank under the accession numbers C HQ204370–HQ204503 (COI) and HQ204504–HQ204543 (16S). F u n d e RESULTS r s A Shell morphology u t h Trochulus hispidus shows higher variation in all shell measurements than the T. oreinos o r M forms, which are at least 0.5 mm smaller (Table 2). Of the four measurements, shell width a and umbilicus width differed between the two T. oreinos taxa. Although the T. oreinos n u s subspecies are smaller and less variable than T. hispidus, ranges overlap. Small specimens of c ri T. hispidus (e.g. those from sample site 17 at Sierningtal-Stixenstein) overlap the range of T. p ts oreinos. Therefore, shell measurements alone are not a suitable discriminating character for the three taxa. Although the systematic measurement error was relatively high (with 1% error probability, from ±0.14 to ±0.18 mm), it did not compromise these results (see Table 2). Measures showed small differences between T. o. oreinos and T. o. scheerpeltzi, but large differences between both taxa and T. hispidus. J Molluscan Stud. Author manuscript; available in PMC 2014 September 04. DUDA et al. Page 7 At first sight T. hispidus has remarkably longer hairs than both T. oreinos subspecies. Hair length of T. hispidus ranges from 0.21 to 0.31 mm (mean 0.27 mm), of T. o. oreinos from 0.03 to 0.09 mm (mean 0.06 mm) and of T. o. scheerpeltzi from 0.04 to 0.08 mm (mean 0.06 mm). Additionally, hairs of the T. oreinos subspecies are often curled or strongly bent, while those of T. hispidus are only slightly bent. The problem with this trait is that elder specimens E and empty shells often lack hairs. For example, of the 118 specimens investigated in T. o. u oreinos, 86 were hairless, but of these individuals 78 were empty shells. Among T. o. r o p scheerpeltzi individuals 59 out of 70 were hairy, most of them collected alive. Among the e P 116 specimens of T. hispidus 106 showed the characteristic long hairs. M C F The T. oreinos taxa show strong development of the lip and aperture (internal rib, basal u tooth, pale around aperture), while in T. hispidus these traits occur only occasionally and n d e rarely in combination (Fig. 4). Most specimens of T. hispidus show only an internal rib or r s A even none of these traits. Both T. oreinos forms consistently show strong irregular riffles, u while only 8 of 116 specimens of T. hispidus have this character. The groove beneath the t h o keel proved not to be a constant character of T. o. scheerpeltzi as two specimens were found r M in which this trait was virtually absent. On the other hand, nine specimens of T. o. oreinos a n were found in which a faint groove was present, five of them from site 34 u s (Tamischbachturm). At this same site three T. o. oreinos with a well-developed groove were c r ip found. t s The occurrence of coarse riffles could not be included in the discriminant analysis, as it is a constant trait of both T. oreinos forms. The variables which most strongly influenced the results of the analysis were the qualitative characters (hair length, groove, internal rib, basal tooth, pale area), especially the apertural traits. The dominance of these factors on the first axis caused a visible ‘horseshoe’ effect (Fig. 5A). The results show a clear differentiation between T. o. scheerpeltzi and T. hispidus. Trochulus o. oreinos and T. hispidus are visibly E u differentiated but still close. The two T. oreinos taxa show an overlap in the discriminant r o analyses (polygons in Fig. 5A) mainly caused by the occurrence of a groove beneath the p e P keel of some specimens of T. o. oreinos. The first axis explains 84% and the second 16% of M the total variation. The first axis separates T. hispidus from the other populations, and C F correlates negatively with the scores associated with the first reciprocal ordering component u (mainly defined by the groove beneath the keel and hair length), and positively with all n d e linear measurements, indicating a general size difference. Trochulus o. scheerpeltzi scores r s A higher on the second axis, which is mainly characterized by a negative correlation with the u scores derived from the second axis of the correspondence analysis (mainly defined by t h o strong development of the groove beneath the keel) (Table 3). Hair length could not be r M analysed in several shells which had obviously lost the hairs (empty shells and some live- a n collected). However, as this is an essential discriminant trait, we included it in our analysis. u s An additional discriminant analysis excluding hair length is shown in Figure 5B. It reveals c r ip the same groups, although T. o. oreinos and T. hispidus are less clearly separated from each t s other. There is one outlier of T. hispidus, a rather small, hairless, empty shell (site 4) which has an internal rib and a pale area around the aperture. This outlier was not included in the J Molluscan Stud. Author manuscript; available in PMC 2014 September 04. DUDA et al. Page 8 computation of the discriminant function, but was subsequently scored to show its position (Fig. 5). Habitat selection and elevation Trochulus o. oreinos was found at elevations from 1,562 to 2,179 m, T. o. scheerpeltzi from E 1,399 to 2,157 m and T. hispidus from 397 to 1,425 m (Table 1). Both T. oreinos taxa were u r found to be restricted to rocky habitats, mostly among sparse alpine grass or in vegetation- o p free areas and mountain pine shrubbery, while T. hispidus preferred moist habitats, in e P particular tall perennial herbs, often near water bodies (Fig. 6). Trochulus hispidus inhabits a M C wider range of vegetation types and landscape structures than both T. oreinos forms. F u n Molecular analysis d e r Among the 132 individuals analysed for COI, 51 different haplotypes were detected. In the s A 38 individuals from which 16S sequence was obtained, 19 different haplotypes were u th observed. In the BI tree based on the COI dataset (Fig. 7) three clearly differentiated groups o r M are found: (1) T. hispidus, (2) T. oreinos scheerpeltzi and (3) T. oreinos oreinos. The same a topology was obtained from both 16S and combined datasets, and with a different tree- n u building algorithm (NJ; data not shown). All three taxa are highly supported (maximum s c r posterior probability) in analyses of all three datasets. The two T. oreinos taxa are well- i p t supported sister groups in all trees. The genetic distances among the three clades are high. s Average p-distances in the COI sequences are 13.3% between the two T. oreinos taxa. Between T. hispidus and T. o. oreinos, and T. o. scheerpeltzi, the values are 14.4% and 16.0%, respectively. The average p-distance for the 16S sequences is 13.7% between the two T. oreinos taxa. Between T. hispidus and T. o. oreinos and T. o. scheerpeltzi the values are 18.0% and 17.4%, respectively. Both T. oreinos subspecies have a somewhat lower haplotype diversity (T. o. scheerpeltzi: 0.88 and T. o. oreinos: 0.70) compared to T. hispidus E (0.95), but the number of sample sites is different in all three (T. hispidus 26, T. o. oreinos 8, u r o T. o. scheerpeltzi, 11). In T. oreinos specimens from one locality had either the same or very p e similar haplotypes, while in T. hispidus quite distinct haplotypes coexist even within one P M locality (e.g. sample site 25). C F u DISCUSSION n d e r The results obtained by the two different approaches (morphological and genetic) agree only s A in some aspects. The clear differentiation between Trochulus hispidus and the two T. oreinos u th taxa is supported by hair and shell morphology, genetic analysis and ecological preferences. o r In contrast, a clear differentiation between T. o. oreinos and T. o. scheerpeltzi is only found M in the genetic analyses, but not in morphology or ecological preferences. a n u s c Differentiation between T. hispidus and T. oreinos r i p t Trochulus o. oreinos and T. o. scheerpeltzi are very similar to each other shell morphology s and ecology, while T. hispidus differs from these taxa in both respects and also appears to be more variable. Hair morphology discriminates the T. oreinos subspecies from T. hispidus, and this character is also useful for juvenile specimens. The basic pattern for hair length and distance between hairs in Trochulus is supposed to be a stable trait as it is determined by J Molluscan Stud. Author manuscript; available in PMC 2014 September 04. DUDA et al. Page 9 glands in the mantle (Kaiser, 1966). Other shell morphological traits also show clear differences between T. oreinos subspecies and T. hispidus. A constant trait of both T. oreinos subspecies are the coarse riffles. Furthermore, only 20% of both T. oreinos subspecies, but nearly 70% of T. hispidus, did not show fully developed apertural traits. Seasonality cannot explain this observation, as the snails were collected at similar times of E the year. A lack of fully developed apertural traits in adult specimens of T. hispidus has been u reported several times in the literature. Weinland (1883) mentioned that it is difficult to find r o p specimens of T. hispidus with a fully developed lip. A similar finding was reported by Geyer e P (1915), who noticed that in Upper Austrian populations of T. hispidus many adult specimens M did not show a lip. C F u Shell measurements alone are not enough for a reliable differentiation. Furthermore, these n d e traits can also be influenced by the environment (Davis, 2004). Our results show that T. r s A hispidus is more variable than both T. oreinos subspecies. Other authors have also described u substantial shell variability in T. hispidus, which can result in difficulties in species t h o recognition (Ložek, 1956; Forcart, 1965; Naggs, 1985; Von Proschwitz, 1993; Proćków r M 2009). However, in contrast to previous studies, we found no specimens with pronounced a n globular shells. u s c r With respect to habitat preferences a clear difference was revealed between T. hispidus and i p ts T. oreinos. Both T. oreinos subspecies are restricted to rocky areas with alpine vegetation at high elevations, whereas T. hispidus extends over a larger range, predominantly in lower areas, preferring moist habitats with a well-developed herb layer, often close to water bodies. The small overlap in the altitudinal distribution of T. hispidus and T. oreinos subspecies might be due to the geomorphological conditions of the northern Calcareous Alps. Springs and creeks usually discharge at the base of the mountain massifs, while plateaus and slopes at high elevation remain dry (Lieb, 1991). Where damp habitats extend E u up to higher elevation, T. hispidus can be found above 1,000 m, as at the sampling sites r o ‘Fischbacher Alpen-Hauereck’ (1,180 m) and ‘Haller Mauern-Arlingsattel’ (1,425 m). The p e P wide distribution of T. hispidus even in formerly glaciated areas of Austria might be due to M the fact that rivers and creeks act as linear corridors along which the snails can easily C F disperse (actively or passively via rafting). The occurrence of T. o. scheerpeltzi at 1,399 m at u the site ‘Hohe Nock-Feichtausee’ is the lowest one ever recorded for this taxon, as the n d e known range is 1,600–2,300 m (Klemm, 1974). Nevertheless, this site fits the habitat r s A preferences of T. o. scheerpeltzi because it is an azonal deep habitat of high alpine u vegetation resulting from the cool microclimate of a sunless shady slope with northern t h o exposure. r M a Our genetic data are in accordance with the morphological and ecological differentiation n u between T. hispidus and T. oreinos. We cannot conclude that T. oreinos and T. hispidus are s c r sister species, owing to our limited sampling of other taxa, and because the phylogenetic i p ts status and monophyly of T. hispidus are unclear (see above). This underlines the need for a complete phylogeny of the genus. The high genetic distances (mean distance for COI 15.2%) (only slightly higher than those between the two T. oreinos subspecies: mean distance for COI 13.3%) suggest that the differentiation between the lineages leading to these two species might have taken place before the Pleistocene glaciations. Also, the J Molluscan Stud. Author manuscript; available in PMC 2014 September 04. DUDA et al. Page 10 difference in the morphological variability in T. oreinos and T. hipidus is reflected in the haplotype variation, T. hispidus showing higher haplotype diversity than the other two taxa. The haplotype diversity found within the T. oreinos taxa is rather low (0.88 and 0.70). Direct comparison of the haplotype diversity is problematic, since the number of sampled localities is higher in T. hispidus than in the two T. oreinos taxa, but it is still meaningful since we E sampled the whole distribution range of the two T. oreinos taxa. Hence, it cannot be u expected that the diversity will increase much by including more localities and samples. r o p Only a part of the distribution range was sampled T. hispidus, but well-differentiated e P haplotypes can be found within a single locality. The low haplotype diversity within the two M T. oreinos taxa is remarkable, since the populations live on isolated mountain peaks. This C F suggests past extinction and recolonization events, perhaps during glacial periods. Other u n studies have reported highly divergent lineages within T. hispidus (Pfenninger et al., 2005; d er Dépraz et al., 2009). However, in our analysis all T. hispidus from the foothills of the s A northeastern Austrian Alps belong to a single haplogroup, distantly related to the u t haplogroups of T. hispidus published so far (Pfenninger et al., 2005, Dépraz et al., 2009). h o r Thus, our analysis reveals yet another lineage that morphologically resembles T. hispidus. M a n Differentiation between T. o. oreinos and T. o. scheerpeltzi u s c While the morphological differentiation between T. hispidus and T. oreinos is r i p t straightforward, the distinction between T. o. oreinos and T. o. scheerpeltzi is difficult, as s there are no apparent differences in hair and shell morphology, size and habitat selection. The only discriminating characters mentioned in the literature are the different geographical ranges and the groove beneath the keel. However, our results indicate that the groove is not a constant trait of T. o. scheerpeltzi as some specimens lack this characteristic, whereas specimens with a groove were found at the western sample sites of T. o. oreinos. The discriminant analysis (Fig. 5) showed overlap of the two taxa. These findings are in E accordance with reports of intermediate forms between the two T. oreinos taxa (Mikula, u ro 1957; Falkner, 1970, 1982) and suggest why the two forms have been classified as p e subspecies. However, from the molecular results there is no indication of hybridization P M between T. o. oreinos and T. o. scheerpeltzi. The distribution of mitochondrial haplotypes is C completely in accordance with the distribution ranges of the two taxa; no haplotypes of T. o. F u oreinos were found in the range of T. o. scheerpeltzi or vice versa. It could be hypothesized n d that the groove is just a result of phenotypic plasticity and/or connected with a yet unknown e rs adaptation to climate or habitat conditions. As the distribution ranges of both subspecies A belong to the same geological formation, it is unlikely that geological factors could be of u t h importance. In this region there is a transition between two local climatic zones – western vs o r M eastern part of the northern margin of the northeastern Alps (as defined by Kilian et al., a 1994). The transition between these two climatic zones covers the eastern part of the n u distribution area of T. o. scheerpeltzi as well as the western part of the T. o. oreinos s c r distribution (Fig. 2), i.e. regions where some morphologically atypical individuals (T. o. i p ts oreinos possessing a groove, T. o. scheerpeltzi lacking a groove) were detected. Subtle climate differences might in some way influence the formation of a partially or fully developed groove. Another explanation for the presence of a groove in the specimens from Tamischbachturm is that they are malformations, although it is hard to explain why this should have happened coincidentally to many individuals. J Molluscan Stud. Author manuscript; available in PMC 2014 September 04.