Inference of evolutionary patterns of the land snail Albinaria in the Aegean archipelago: Is vicariance enough?

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1 Molecular Phylogenetics and Evolution 44 (2007) Inference of evolutionary patterns of the land snail Albinaria in the Aegean archipelago: Is vicariance enough? Vassilis Douris a,1, Sinos Giokas b, Diogo Thomaz a, Rena Lecanidou a, George C. Rodakis a, a National and Kapodistrian University of Athens, Department of Biochemistry and Molecular Biology, Panepistimioupolis, Athens, Greece b University of Patras, Department of Animal Biology, Patras, Greece Received 23 October 2006; revised 13 December 2006; accepted 3 January 2007 Available online 19 January 2007 Abstract Mitochondrial DNA sequences from 16S rrna and ATPase8 genes were used to investigate phylogeographic patterns of the land snail Albinaria (Gastropoda: Clausiliidae) in the Aegean archipelago. Forty-two populations of Albinaria were analyzed, mainly A. turrita, A. caerulea and A. brevicollis, collected from 22 Aegean islands and certain surrounding regions. Maximum parsimony, maximum likelihood and Bayesian analyses on 16S rrna and combined datasets produced trees that share signiwcant similarity and reveal a phylogeny with distinct branches which are in general, but not full, agreement with current taxonomy. The Aegean taxa are not monophyletic as a whole, since A. turrita does not cluster with A. caerulea and A. brevicollis. The latter form a distinct monophyletic cluster, within which two groups are evident. These groups do not readily correspond to currently accepted morphospecies; one contains the populations that inhabit the central part of the archipelago plus some eastern islands, while the other contains populations whose geographic distribution is restricted to the southeastern part of the archipelago. The divergence between these two groups is attributed to vicariance events that primarily shape contemporary distributions. Although dispersal may also be present, certain small- and large-scale vicariance events can be traced; alternative phylogeographic hypotheses are discussed in view of the historical biogeography of the region Elsevier Inc. All rights reserved. Keywords: Albinaria; Land snails; Phylogeography; Aegean palaeogeography; 16S rrna; ATPase8; Vicariance; Dispersal 1. Introduction * Corresponding author. address: grodakis@biol.uoa.gr (G.C. Rodakis). 1 Present address: National Center for ScientiWc Research Demokritos, Institute of Biology, P.O. Box 60228, Aghia Paraskevi, Athens, Greece. The Aegean archipelago (Fig. 1) is an area especially suited for phylogeographic studies because it consists of numerous small and larger islands inhabited by a high number of taxa with comparatively well-known distribution (Hausdorf and Hennig, 2005). Several works suggest that the distributional patterns of the studied groups in that area may rexect palaeogeographical patterns or historical events; these groups include amphibians (Beerli et al., 1996), reptiles (Kasapidis et al., 2005; Poulakakis et al., 2005), isopods (Sfenthourakis, 1996), beetles (Fattorini, 2002a,b; Chatzimanolis et al., 2003), butterxies (Dennis et al., 2000), scorpions (Gantenbein and Larqiadèr, 2003; Gantenbein and Keightley, 2004; Parmakelis et al., 2006) and land snails (Douris et al., 1995, 1998a; Welter- Schultes and Williams, 1999; van Moorsel, 2001; Parmakelis et al., 2005). However, variable diverentiation patterns may rexect diverential intrinsic response of each group to the historical events. Therefore, there are still unanswered questions about the intrinsic properties of each group, as well as about the role of vicariance and dispersal in shaping current distributional patterns in the archipelago. The present day Aegean archipelago has been shaped by the diverential evects of tectonism, volcanism and eustatism. Summarizing these palaeo-processes (Fig. 2), four main stages in the palaeogeographic evolution of the Aegean region can be distinguished. During the Wrst stage (upper and middle Miocene, MYA) there was the /$ - see front matter 2007 Elsevier Inc. All rights reserved. doi: /j.ympev

2 V. Douris et al. / Molecular Phylogenetics and Evolution 44 (2007) Fig. 1. Distribution of genus Albinaria (small map; modiwed from Douris et al., 1998b) and collection sites of the samples used in this study at the Aegean archipelago and Greek mainland (large map). DiVerent symbols are used to denote diverent species; numbers 1 44 correspond to the relevant numbers of Table 1. The dashed rectangle in the small map indicates the area shown in the large map. continuous landmass of the Agäis (Dermitzakis and Papanikolaou, 1981). The second stage (late Seravallian to early Tortonian, 12 5 MYA) involves a rough segregation of the Agäis due to the slow formation of a sea channel (mid- Aegean trench), beginning at the end of the middle Miocene (12 MYA) and fully completed during the early late Miocene (10 9 MYA) (Creutzburg, 1963; Dermitzakis and Papanikolaou, 1981). This resulted in the separation between west Aegean and east Aegean islands. In the Messinian (6 5.3 MYA) the Mediterranean basin dried up, as a result of the closing of the Strait of Gibraltar (Krijgsman et al., 1999) and overland migration between islands and from the mainland was possible. About 5.3 MYA, the Strait of Gibraltar reopened (Beerli et al., 1996; Krijgsman et al., 1999; Duggen et al., 2003) and within 1000 years the basin was rewlled from the Atlantic Ocean, and the mid-aegean trench became permanent. During the third stage (5 2 MYA), further sea expansion and landmass compartmentalization took place. The south Cycladic plateau formed an insular landmass, which was isolated from the north Cyclades by a seaway during Pliocene and upper Pleistocene ( MYA) (Anastassakis and Dermitzakis, 1990) and Rhodes was isolated from Asia Minor on late Pliocene or on early Pleistocene. However, the land connection between northern Dodecanese islands and their respective opposite coasts of Asia Minor was retained till the beginning of the Holocene. Finally, the fourth stage (during the Pleistocene) involved intense eustatic and climatic changes. In the Pleistocene, all present day islands were in the same position as present (Perissoratis and Conispoliatis, 2003); however, during the glacial maxima, the sea-level was as much as 200 m lower than today (Beerli et al., 1996). It is not clear to what extend these processes are rexected on contemporary distributions of diverent taxa. Among several studied groups, land snails are particularly useful model organisms for the evaluation of diverent phylogeographic hypotheses because of their limited dispersal abilities (Pfenninger et al., 1996; Giokas and Mylonas, 2004; Parmakelis and Mylonas, 2004). One of the most well studied, though a still largely not well-understood model is the pulmonate land snail Albinaria (Pulmonata, Alopiinae, Clausiliidae). Albinaria is the most speciose genus within the family of Clausiliidae (see small map in Fig. 1 for its distribution) containing about 400 species and subspecies (Gittenberger, 1998; Nordsieck, 1999) and has served as a model for studies of ecological and morphological diverentiation, molecular evolution, and phylogeography. Nevertheless, many aspects remain uncertain, given the large number of taxa whose classiwcation has not yet been evaluated with solid synapomorphic characters. A number of

3 1226 V. Douris et al. / Molecular Phylogenetics and Evolution 44 (2007) Fig. 2. Palaeogeographic maps of Greece from Miocene to present (redrawn after Creutzburg, 1963; Dermitzakis and Papanikolaou, 1981; Dermitzakis, 1990, and modiwed after permission from Parmakelis et al., 2005). studies within the last decade have tackled speciwc issues of the Alopiinae subfamily phylogeny, including Albinaria, based mainly on mitochondrial (16S and 12S rrna, cytochrome oxidase subunit I) and nuclear DNA sequences (ITS: rrna Internal Transcribed Spacers) (Schilthuizen et al., 1995, 2004; Douris et al., 1995, 1998a,b; van Moorsel et al., 2000; van Moorsel, 2001; Uit de Weerd et al., 2004; Uit de Weerd and Gittenberger, 2005). These studies have signiwcantly challenged conventional taxonomy, and have shed light on certain aspects of Albinaria molecular evolution and biogeography. Most of these studies include Albinaria taxa from Crete, the Greek mainland, the western coast of Turkey and the eastern Mediterranean. As far as the Aegean archipelago is concerned, there are species with almost exclusively Aegean distribution that have drawn relatively little attention in the literature so far: A. turrita has a limited distribution in the western arc of the Cyclades, the islands of Kea, Sifnos and Milos. A. caerulea is distributed in the rest of the Cyclades and parts of coastal West Turkey (though there is an isolated colony of A. caerulea on the Greek mainland, around the ancient temple of Artemis in Vravrona, Attica), while A. brevicollis is found in coastal West Turkey, in certain islands of the East Cyclades and mainly in the Dodecanese islands. These three species are the only ones found in the Cyclades and in most of the Dodecanese islands (except for A. lerossiensis in Kos and Leros, A. munda in Kos and West Turkey and A. klemmi in Rhodes, which are found near, but not syntopically, with A. brevicollis populations). Questions on the status of certain taxa were raised using cross-breeding and allozyme data (Mylonas et al., 1987; Ayoutanti et al., 1987, 1993). A comparative study of morphological and mitochondrial DNA restriction site polymorphisms (Douris et al., 1995) has provided some information on the intraspeciwc diverentiation of some Aegean taxa, mainly A. turrita and A. caerulea, but could not accurately depict population variability or eyciently resolve phylogenetic relations at the interspeciwc level. For example, in the phylogeny of Douris et al. (1995), all A. turrita and A. caerulea populations cluster within a single monophyletic group; this could imply a closer relation and a potential common descent for all Aegean taxa. However, analysis of later available 16S rrna data that included also many Cretan and mainland species (Douris et al., 1998a) did not support any close relation between A. turrita and A. caerulea, which clustered with diverent, distant groups. In general, the phylogeny and evolutionary dynamics of Albinaria populations from the Aegean islands have

4 V. Douris et al. / Molecular Phylogenetics and Evolution 44 (2007) not yet been studied in detail with the use of molecular markers. Such an approach would be valuable for inferring the evolutionary processes of these species in the Aegean area. In order to examine whether present day distributions are shaped by vicariance, the phylogeny of extant Aegean Albinaria populations needs to be correlated with the available information on the complex geological history of the Aegean archipelago. In this study, we report the result of a comprehensive phylogenetic analysis encompassing samples from 42 diverent collection sites (38 sites on 22 islands and islets of the Aegean archipelago and four sites on the Greek mainland), which represent the three major Aegean species as well as certain outgroup mainland taxa, with the use of DNA sequences from two mitochondrial genes, 16S rrna and ATPase8, a fast marker that has been introduced recently for population structure analysis in land snails (Goodacre et al., 2006). The phylogenetic relevance of current taxonomy for Aegean populations is examined and alternative phylogeographic hypotheses on the distribution and evolutionary history of the Aegean Albinaria taxa are discussed in view of the historical biogeography of the region. 2. Materials and methods 2.1. Sampling, coding, selection and designation of ingroup and outgroup taxa Specimens from 42 Albinaria populations were collected from restricted areas (no more than 20 square meters) in 22 islands and islets of the Aegean archipelago and Wve localities in the Greek mainland (Fig. 1). The species and collecting sites are shown in Table 1. Species designation, prior to our analyses, was based on morphological diagnostic characters (Nordsieck, 1999) despite the problems associated with this typological approach. On these grounds, A. brevicollis, which has been considered as a member of the so-called caerulea subgroup (Nordsieck, 1999), distributed in an adjacent area to that of A. caerulea, was treated as a diverent species. Given the relatively large sampling area, each specimen was assigned a distinct code (introduced initially in Douris et al., 1998b), where the Wrst three letters represent the species name, while the rest represent the sampling location and the individual sample within the population. All codes are listed in Table 1. The Aegean populations under study belong to the three major Aegean species, namely A. turrita, A. caerulea and A. brevicollis. An A. discolor sample from the islet of Velopoula was used as the most proximal representative of Greek mainland species and available sequences of A. discolor from Monemvassia, Peloponnese, were used for comparison. An A. grisea population from Ymittos, Attica, was included since it was used as an outgroup in a previous study (Douris et al., 1995), as well as an A. butoti population from the Peloponnese, which together with A. grisea appears as a sister group to A. caerulea in a previous analysis of 16S rrna (see Fig. 4 of Douris et al., 1998a). The 16S rrna sequences of Isabellaria saxicola and Sericata sericata (Douris et al., 1998b) were used as more distant outgroups, according to former phylogenetic analyses of the subfamily Alopiinae (Douris et al., 1998b; van Moorsel, 2001; Uit de Weerd and Gittenberger, 2005) DNA extraction, PCR ampliwcation and sequencing Total DNA was extracted from the foot of each animal by SDS/Proteinase K treatment and subsequent phenol extraction as previously described (Douris et al., 1998b). DNA concentration was determined spectrophotometrically. Primers for the ampliwcation reactions of 16S rrna were used as previously described (Douris et al., 1998a,b), in order to amplify a region corresponding to bases of the A. caerulea mitochondrial genome (Hatzoglou et al., 1995). For the ampliwcation of the ATPase8 gene, primers LEU (5 -ACACCAATAGAAAACTATAAAG-3 ) and ASN (5 -CATCCCTCTAAGTAGAGCTTGA-3 ) were designed after comparison of the relevant sequences for A. turrita (Lecanidou et al., 1994) and A. caerulea (Hatzoglou et al., 1995), which are the only available sequences for this mtdna segment in Albinaria. A fragment of mtdna, corresponding to bases of the A. caerulea mitochondrial genome, was ampliwed; this includes the entire ATPase8 gene as well as part of the genes coding for trna Asn and trna Leu(UUR). AmpliWcation was carried out as previously described (Douris et al., 1998b). Sequences were obtained by directly sequencing the PCR products using the Sequenase v.2 PCR product sequencing kit (Amersham), with the primers used for the ampliwcation reaction serving also as sequencing primers. Sequences were deposited in GenBank (accession numbers shown in Table 1) Alignment of sequences and test for saturation and partition homogeneity Two datasets were used; one containing 47 aligned 16S rrna sequences (common length 401 bp) of A. caerulea, A. brevicollis, A. turrita, A. discolor, A. grisea, A. butoti, as well as of the outgroup taxa I. saxicola and S. sericata, and a second one containing 40 ingroup taxa for which both 16S rrna and corresponding ATPase8 sequences were available (common length of the concatenated dataset 568 bp) of A. caerulea, A. brevicollis, A. turrita. Sequences from the trnas Xanking the ATPase8 gene were not included in the analysis because they were not available for all taxa and they should be treated as separate markers with diverent substitution models and evolutionary constraints. Given the low phylogenetic signal of these regions, only the full-length ATPase8 gene was included in the analysis. The sequences were aligned with CLUSTALX (Thompson et al., 1997) using the default alignment parameters and then adjusted manually. Loop regions were unambiguously aligned, with minimal gap insertions. For the ATPase8 dataset, the amino acid sequence alignment served as template for subsequent nucleotide sequence alignment. The

5 1228 V. Douris et al. / Molecular Phylogenetics and Evolution 44 (2007) Table 1 Codes, species, collection localities (see also Fig. 1) and GenBank sequence accession numbers of the specimens sampled No Code Species Locality Individuals sequenced 16S rrna ATPase8 1 bre.anh1 A. brevicollis AnaW island (Chora) 2 DQ DQ DQ DQ bre.anm1 A. brevicollis AnaW island (Monastery, site 1) 1 DQ DQ bre.anm2 A. brevicollis AnaW island (Monastery, site 2) 1 DQ DQ bre.ast1 A. brevicollis Astypalea island (Chora) 2 DQ N/A DQ bre.hal1 A. brevicollis Chalki island (Castle) 1 DQ DQ bre.kan1 A. brevicollis Kandeliousa islet 1 DQ DQ bre.kos2 A. brevicollis Kos island (Chora) 1 DQ DQ bre.rod1 A. brevicollis Rhodes island (500 m E of Port) 1 DQ DQ bre.rod2 A. brevicollis Rhodes island (Port) 1 DQ DQ bre.til1 A. brevicollis Tilos island (Livadia) 1 DQ DQ bre.til2 A. brevicollis Tilos island (Road from Megalo Chorio to Castle) 1 DQ DQ cae.amo1 A. caerulea Amorgos island (Chora) 3 X83390 X83390 DQ DQ DQ DQ cae.and1 A. caerulea Andros island (Kaparia) 2 DQ DQ DQ DQ cae.apr1 A. caerulea Andiparos island (just out of the port) 1 DQ DQ cae.apr2 A. caerulea Andiparos island (close to the Cave) 1 DQ DQ cae.fol1 A. caerulea Folegandros island, E of Misotrouli cape (Ribbed form) 1 DQ DQ cae.fol2 A. caerulea Folegandros island, Misotrouli cape (Semi-ribbed form) 1 DQ DQ cae.fol3 A. caerulea Folegandros island, W of Misotrouli cape (Smooth form) 1 DQ DQ cae.hra1 A. caerulea Iraklia island 1 DQ DQ cae.nax1 A. caerulea Naxos island (Myloi) 1 DQ DQ cae.nax2 A. caerulea Naxos island (Fotodoti Monastery, 800 m height) 1 DQ DQ cae.nax3 A. caerulea Naxos island (Mt Zas, 1000 m height) 1 DQ DQ cae.par1 A. caerulea Paros island, (road from Lefkes to Agioi Pantes) 1 DQ DQ cae.par2 A. caerulea Paros island (Marble mines at Marathi) 1 DQ DQ cae.sik3 A. caerulea Sikinos island (Ai-Giorgis) 1 DQ DQ cae.sik4 A. caerulea Sikinos island (Zoodohos Pigi Monastery) 1 DQ DQ cae.syr1 A. caerulea Syros island (Galissas bay) 1 DQ DQ cae.syr2 A. caerulea Syros island (Ano Syros) 1 DQ DQ cae.thi1 A. caerulea Thira island (Prophitis Ilias) 1 DQ DQ cae.tin1 A. caerulea Tinos island (Hill N to Megalochari) 1 DQ DQ cae.tin2 A. caerulea Tinos island (S of Megalochari) 1 DQ DQ cae.vra1 A. caerulea Vravrona, Attica (Sanctuary of Artemis) 1 DQ DQ tur.kea1 A. turrita Kea island (Panagia Kastriani) 1 DQ DQ tur.kea2 A. turrita Kea island (Poisses) 1 DQ DQ tur.kea4 A. turrita Kea island (Poisses, South Xank of beach) 1 DQ DQ tur.mil1 A. turrita Milos island (Provatas) 1 DQ DQ tur.sif1 A. turrita Sifnos island (Kamares bay) 3 DQ DQ DQ DQ DQ DQ tur.sif2 A. turrita Sifnos island (Vathi bay) 1 DQ DQ dis.mon2 A. discolor Monemvassia, Peloponnese 1 AF N/A 40 dis.vel1 A. discolor Velopoula islet 1 DQ N/A 41 gri.ymi1 A. grisea Mt Ymittos at Kesariani, Attica 1 AF N/A 42 but.ksm1 A. butoti Geraki (2 km to Kosmas), Lakonia, Peloponnese 1 AF N/A 43 Sse.Ste1 Sericata sericata Steni DirWs, Evia 1 AF N/A 44 Isa.Ymi1 Isabellaria saxicola Mt Ymittos at Kesariani, Attica 1 AF N/A Note: N/A, not available. aligned sequences of both 16S rrna and ATPase8 are available from the authors upon request. We examined saturation of phylogenetic information in the datasets of 16S RNA and ATPase8, using the Xia s test employed in DAMBE (Xia, 2000; Xia and Xie, 2001). The sequences of the two genes are linked and thus can be combined in a single analysis to increase the information content. However, in order to further examine the validity of the concatenation, the LRT partition-homogeneity test, also known as incongruence-length diverence test (Farris et al., 1995), was run in PAUP v.4b10 (SwoVord, 2002) using 1000 repartitions. This indicated that there is no signiwcant conxict between the datasets (p D 0.68) Phylogenetic analyses Phylogenetic analyses were performed using maximum parsimony (MP), maximum likelihood (ML) and Bayesian

6 V. Douris et al. / Molecular Phylogenetics and Evolution 44 (2007) inference (BI). MP analysis of the 16S rrna, ATPase8 and concatenated datasets was performed using PAUP 4.0b10. Characters were unordered with equal weight and gaps were treated as missing. Heuristic searches were performed using stepwise addition and tree-bisection-reconnection (TBR) branch-swapping (SwoVord et al., 1996). ConWdence in the nodes was assessed by 1000 bootstrap replicates with random addition of taxa (Felsenstein, 1985). The most appropriate model of DNA substitution and the parameter estimates for tree construction in the ML analysis was selected with MODELTEST 3.7 (Posada and Crandall, 1998), according to the Akaike information criterion (AIC; Akaike, 1974). For the 16S RNA dataset of the 47 taxa the best-wt model, was TVM+I+G (Rodriguez et al., 1990). For the 40 taxa, the best-wt models were TVM+G (Posada and Crandall, 1998) for the 16S RNA, TrN+G (Tamura and Nei, 1993) for ATPase8, and K81uf+G (Posada and Crandall, 1998) for the concatenated dataset. ML searches were performed with PHYML v2.4.4 (Guindon and Gascuel, 2003) and were evaluated using 500 bootstrap replicates. Bayesian analysis (BI) was performed with MrBayes 3.1 (Huelsenbeck and Ronquist, 2001) using the parameters of the substitution models that were proposed by the AIC in MODELTEST (see above). In the concatenated dataset, site-speciwc rate variation partitioned by gene was allowed, applying in each gene partition the substitution model suggested by MODELTEST according to the AIC. Four Metropolis-coupled chains were run for generations. A tree was sampled every 100th generation. A test for stationarity of likelihood values was carried out by examining the average standard deviation of split frequencies of the two simultaneous and independent runs (convergence diagnostic, see MrBayes 3.1 manual). Stationarity was achieved before generations. After reaching stationarity, the Wrst 2500 trees (burnin 25%) of each run were discarded, from the sample of 20,002 trees (of the two runs). Then a 50% majority-rule consensus tree, with posterior probabilities, was constructed from the remaining 15,002 trees. To compare topologies yielded by MP, ML and BI searches as well as enforced alternative topologies (i.e. A. caerulea and A. brevicollis forming two reciprocally monophyletic clades, and A. turrita forming a monophyletic clade with the caerulea brevicollis clade), the approximately unbiased (AU) and Shimodaira Hasegawa (SH) tests implemented in CONSEL (Shimodaira and Hasegawa, 2001) was used. Site-wise log-likelihoods for all trees, for both the 16s and the concatenated datasets, were estimated using PAUP and used as input for CONSEL Test for molecular clock and estimation of divergence times A molecular-clock likelihood-ratio test (LRT) (Huelsenbeck and Crandall, 1997) was performed with PAUP to determine if there is statistical diverence in evolutionary rates among clades. The results of the LRT test indicated that our sequences do not conform to a clocklike evolution (see Section 3). Therefore, time divergence estimates were obtained by relaxing the molecular clock assumption, and implementing the nonparametric rate smoothing (NPRS) method of Sanderson (1997). Two alternative candidate vicariance events were used at the same node (see Fig. 3): the formation of the mid-aegean channel (12 9 MYA), and the permanent separation of the areas across this channel after the end of Messinian salinity crisis (6 4 MYA). That particular node has been selected as a calibration point because it is the node where the taxa of the A. caerulea A. brevicollis group that are distributed east and west of the mid-aegean channel are split. The divergence times (and the corresponding 95% conwdence limits) of the respective nodes for the two calibration events were estimated according to the suggestions described in the r8s manual ( ginger.ucdavis.edu/r8s), enforcing the constraint command (since the minimum and maximum ages were used), employing 100 bootstrapped phylograms produced under the ML criterion using the same parameters as in the original ML analysis. The reliability of the applied method (NPRS) to explain the branch length variation was further explored using the cross-validation option (Sanderson, 2003) for the Bayesian inference 50% majority-rule consensus tree derived from the 16S RNA dataset. 3. Results In total, 51 specimens were sequenced for 16S rrna and from these, 43 were sequenced for ATPase8 (Table 1). No ampliwed DNA product was obtained for the ATPase8 gene in the two A. brevicollis from Astypalea, A. grisea, A. butoti, and A. discolor, as well as the two outgroup taxa (genera Isabellaria and Sericata), most probably due to mismatch of the primers to mtdna templates. Certain specimens sequences were identical to each other, either in the 16S rrna gene (bre.ast1.1 and bre.ast1.2) or in both genes examined (tur.sif1.1 and tur.sif1.2, as well as bre.anh1.1 and bre.anh1.2). A case of special interest was found in Folegandros, where we sampled A. caerulea individuals with strikingly diverent shell morphology ( smooth, cae.fol3; ribbed, cae.fol1; semi-ribbed, cae.fol2), which were traditionally considered as diverent subspecies (Nordsieck, 1999). It is interesting that the specimens sequenced, despite their diverent shell morphology, have identical (cae.fol1 and cae.fol3) or very similar (cae.fol2) nucleotide sequence for both ampliwed fragments. All the aforementioned cases of sequence identity concern individuals obtained from the same collection site. This is not surprising, given that preliminary data from single-strand conformation polymorphisms of the ATPase8 gene (Thomaz, Douris, Rodakis and Lecanidou, unpublished results) indicate that although populations are expected to be highly structured, individuals collected from small areas (demes) most frequently appear monomorphic. In order to minimize computation times, in cases of

7 1230 V. Douris et al. / Molecular Phylogenetics and Evolution 44 (2007) Fig. 3. Bayesian inference 50% majority-rule consensus trees of 16S rrna dataset for 47 taxa (left) and concatenated (16S rrna plus ATPase8) dataset for 40 taxa (right). Nodes with posterior probability >0.7 are indicated as white circles. Posterior probabilities (100 ) are shown next to nodes. The numbers in parentheses indicate percent bootstrap support after 500 maximum-likelihood replicates; the asterisk indicates a node where the ML topology is diverent and the bootstrap support is missing. Boxes A and B (dashed) indicate relevant groups (see text), while gray areas indicate subgroups within group A (light gray, a1; medium gray, a2+; dark gray, a3). The arrow at the 16S RNA tree indicates the node used as reference node for NPRS. Bars below trees are branch length indices. sequence identity only one sequence per collection site was used in the analysis. Thus, 47 diverent 16S rrna and 40 diverent ATPase8 sequences were subjected to further analysis for tree construction. Alignment of the 47 diverent 16S rrna sequences resulted in a dataset with 401 total characters (including gaps). From these, 138 were variable and 103 were parsimony-informative. MP, ML and BI analyses were performed (trees provided as Supplementary material). Equally weighted MP analysis produced 1574 equally most-parsimonious trees (lengthd 367, HI D0.400, RI D0.813). Maximum-likelihood analysis, under the TVM+I+G model, resulted in a tree with lnl D The majority-rule tree for the Bayesian inference method and the posterior probabilities for its nodes are shown in Fig. 3, as well as the bootstrap support for the relevant nodes in the ML analysis. Topologies yielded by MP, ML and BI searches were not diverent at the 5% level (Table 2). As shown in Fig. 3, all Table 2 Comparisons of alternative topologies using likelihood based methods Dataset Trees Rank ln[l] AU SH Possible 16S RNA BI Yes ML Yes MP Yes C p < No C p < No Concatenated BI Yes ML Yes MP Yes C p < No Results are listed for the topologies tested. BI, Bayesian inference; ML, maximum likelihood; MP, maximum parsimony; C1, A. turrita forming a monophyletic clade with the caerulea brevicollis clade; C2, A. caerulea and A. brevicollis forming two reciprocally monophyletic clades; AU, approximately unbiased test; SH, Shimodaira Hasegawa test.

8 V. Douris et al. / Molecular Phylogenetics and Evolution 44 (2007) nominal Albinaria taxa appear monophyletic, well dewned from the outgroup taxa. As far as the ingroup taxa are concerned, Wve major clades are formed, supported with high bootstrap values and posterior probability indices. These clades either correspond to species already dewned by current taxonomy (A. turrita, A. discolor) or to species groups whose close relation has been reported by previous studies (A. grisea with A. butoti, A. caerulea with A. brevicollis). DiVerent phylogenetic methods employed yielded statistically undistinguishable trees (Table 2), in which the ingroup clades exhibit certain common features: A. turrita is always the sister group to all other Albinaria taxa in the analysis, while the node corresponding to the most recent common ancestor of A. caerulea and A. brevicollis is always derived relative to the most recent common ancestor of this and the group of A. discolor and A. grisea A. butoti. This indicates that A. turrita is derived from a diverent lineage than A. caerulea and A. brevicollis. An alternative hypothesis of Aegean taxa single common descent (i.e. A. turrita A. caerulea A. brevicollis forming a single monophyletic clade) was examined but the enforced constraint topology was rejected (Table 2). All analyses indicate that A. caerulea and A. brevicollis belong to a single monophyletic cluster, at least with respect to the taxa included in this study. A major dichotomy among the taxa forming this cluster is evident, supported by high bootstrap values and posterior probability (Fig. 3). Interestingly, this dichotomy does not readily correspond to a division between A. caerulea and A. brevicollis morphospecies. One group (termed hereafter group A) contains all A. caerulea and certain A. brevicollis specimens (from Kos, Tilos, AnaW and Kandeliousa), while a second group (group B) contains the remaining A. brevicollis (from Rhodes, Tilos, AnaW, Chalki and Astypalea). Within group A, certain subgroups with signiwcant similarity (or even identity) among their members are also evident (Fig. 3). Subgroup a1 contains all the A. caerulea representatives from the islands Andros, Tinos and Antiparos, as well as A. brevicollis from Kos. Subgroup a2 contains A. caerulea from Amorgos, Iraklia, Paros and Syros, as well as A. brevicollis from Kandeliousa and Tilos. Subgroup a3 contains only A. caerulea from the islands Thira, Sikinos and Folegandros, as well as the mainland colony of A. caerulea in Vravrona, Attica. Finally, the rest of A. caerulea from Naxos, Paros, Syros and A. brevicollis from AnaW could not be ayliated to any subgroup or form groups themselves with some signiwcant support. In order to further investigate the relations within and between the major taxa distributed in the Aegean (A. turrita, A. caerulea and A. brevicollis), the analysis was expanded to incorporate ATPase8, and a concatenated (16S rrna plus ATPase8) dataset was generated for 40 taxa. From the 568 total characters (397 16S RNA and 171 ATPase8 sites), 190 were variable (91 16S rrna and 99 ATPase8), from which 160 were parsimony-informative (75 16S rrna and 85 ATPase8). MP, ML and BI analyses were performed (trees provided as Supplementary material). Equally weighted MP analysis produced 312 equally most-parsimonious trees (length D 380, HI D 0.324, RI D 0.891). Maximum-likelihood analysis, under the K81uf+G model, resulted in a tree with lnl D The majority-rule tree that resulted from the Bayesian inference method and its node posterior probabilities are shown in Fig. 3, as well as the bootstrap support for the relevant nodes in the ML analysis. Topologies yielded by MP, ML and BI searches were not divering at the 5% level (Table 2). In the unrooted phylogenies (Fig. 3), A. turrita taxa are depicted as potential outgroups, given the basal position of A. turrita compared to A. caerulea A. brevicollis in the 16S rrna phylogeny (Fig. 3). The major subdivisions evident in the 16S rrna trees are evident here as well (strong support for A. turrita, group A and group B clades). An alternative morphospecies-monophyly hypothesis (i.e. A. brevicollis and A. caerulea forming two reciprocally monophyletic clades) was examined, but the enforced topology was rejected both for the 16S RNA and the concatenated datasets (Table 2). The resolution close to the terminal nodes is now much improved compared to the 16S rrna data alone. Thus, within the A. turrita clade, a single common origin of all the populations from Kea gains more support, while the populations from Sifnos appear more diverentiated. Within group B, the branching order of the taxa included is now almost fully resolved. Finally, within group A all the subgroups dewned through the 16S rrna data alone (a1, a2 and a3) are conwrmed after the addition of the ATPase8 sequences, but most of the taxa previously unrelated to any subgroup, now cluster together with subgroup a2 taxa in a new broader subgroup (termed subgroup a2+, Fig. 3). This latter subgroup encompasses specimens of A. caerulea from Naxos, Paros, Amorgos, Iraklia and Syros, as well as A. brevicollis from Kandeliousa, Tilos and AnaW, leaving only one taxon (coe.syr2.1) without clear ayliation. It is noteworthy that, with the exception of the A. brevicollis specimens included in group A, subgroups a1, a2+ and a3 contain representatives from populations of close geographical proximity (Fig. 4). The branching order among the three subgroups and coe.syr2.1 cannot be readily derived from the available data. The likelihood-ratio test rejected the hypothesis of a homogenous clocklike rate for the tree produced by the 33 sequences of A. caerulea A. brevicollis (LRT D , d.f. D 31, χ 2 < 0.005). Homogenous clocklike rate was rejected in all datasets (results not shown). This suggests that the genetic distances between populations cannot be readily used in order to estimate a local rate of evolution for these clades. However, time divergence estimates were obtained by relaxing the molecular clock assumption, and implementing the nonparametric rate smoothing (NPRS) method of Sanderson (1997). These divergence times (and the corresponding 95% conwdence limits) of the respective nodes for the two alternative calibration events are presented in Table 3.

9 1232 V. Douris et al. / Molecular Phylogenetics and Evolution 44 (2007) Fig. 4. Geographic distribution of the Aegean groups and subgroups identiwed by the phylogenetic analysis of the concatenated dataset (Fig. 3). t, A. turrita group; A and B, groups of A. caerulea A. brevicollis; a1, a2+ and a3, subgroups within group A. The arrows indicate possible dispersal routes of populations bearing a1 or a2+ haplotypes either from the central Aegean or from unidentiwed areas (denoted by question mark) in West Turkey. Table 3 Estimated ages in MYA and the correspondent 95% conwdence limits in parenthesis for selected nodes, according to the two calibrations for node A vs. B, obtained using the NPRS method Phylogenetic separation event (nodes) 4. Discussion Age Calibration at 4 6 MYA Calibration at 9 12 MYA a1 vs. a2 2.5 (1.4 4) 4.9 (2.8 8) (a1 + a2) vs. a3 2.9 (2 4) 5.8 (4 8) (A + B) vs. grisea-discolor 25 ( ) 48.5 ( ) (A+B+grisea-discolor) vs. (turrita) 55 ( ) ( ) The revealed molecular phylogeny is at odds in several aspects with current taxonomy in Albinaria (Nordsieck, 1999) where species dewnitions are typological and based on a combination of morphological nondiagnostic characters that do not provide unambiguous synapomorphies for monophyly. Such incongruence between molecular and typological delimitations has been repeatedly found in Albinaria (Douris et al., 1998b; Uit de Weerd et al., 2004; Uit de Weerd and Gittenberger, 2005) urging the use of molecular phylogenetic analyses. On these grounds, A. brevicollis has been considered as a member of the so-called caerulea subgroup, distributed in an adjacent area to that of A. caerulea, but it was treated as a diverent species (Nordsieck, 1999). However, maximum parsimony analysis of morphological characters disputed the monophyly of A. brevicollis, and in the revealed phylogeny A. caerulea and A. brevicollis form a monophyletic group and A. brevicollis appears paraphyletic (Giokas, 1996, 2000). Phylogenetic analyses that used nuclear markers (ITS1 and ITS2, van Moorsel, 2001) or allozymes (Ayoutanti et al., 1993) support this result. The aforementioned MP analysis of morphological characters showed that the two forms of A. caerulea (ribbed and smooth) on Folegandros island that have the same mtdna sequence, are members a broader, partly unresolved monophyletic group, that includes all the populations of A. caerulea and A. brevicollis in the Aegean archipelago, and asserted that shell ribbing is a highly homoplasious character. Similarly, on morphological grounds A. turrita was considered by Nordsieck (1999) as a member of the so-called caerulea subgroup. In a previous study (Douris et al., 1995) we had also suggested that A. caerulea and A. turrita do form a monophyletic group. In that paper, we supported a scenario that included some kind of monophyletic origin for the central Aegean taxa (A. turrita and A. caerulea). However, in the present analyses of 16S rrna, A. turrita always occupies the most basal position, while A. caerulea with A. brevicollis the most derived, with respect to the mainland taxa (A. discolor, A. grisea A. butoti) included in the analyses, and the alternative hypothesis of A. turrita A. caerulea A. brevicollis monophyly was rejected (Table 2). This result is in concordance with an earlier phylogenetic analysis using allozymes (Ayoutanti et al., 1993) as

10 V. Douris et al. / Molecular Phylogenetics and Evolution 44 (2007) well as with the analysis of a limited number of representative taxa with cytochrome oxidase subunit II sequences (Douris, 1997). These Wndings call for a re-evaluation of the major typological clades in Albinaria. More important, they raise the question of how A. turrita and A. caerulea reside in such geographical proximity in the Aegean region if not through vicariance. Several studies (Douris et al., 1995, 1998a; Beerli et al., 1996; Chatzimanolis et al., 2003; Parmakelis et al., 2003, 2005; Poulakakis et al., 2003, 2005; Kasapidis et al., 2005), suggest that the present distribution of most of the terrestrial Aegean taxa, is mainly shaped through vicariant phenomena resulting from the complex geological history of the east Mediterranean and especially of the Aegean archipelago. Nevertheless, dispersal over sea or through occasional land bridge corridors is also involved (see Douris et al., 1998a; Dennis et al., 2000; Kasapidis et al., 2005). As for the A. caerulea A. brevicollis clade, the absence of taxa from the Greek mainland (except for one spot occurrence at the archaeological site of Vravrona) and from the west Cyclades, imply an eastern origin for the cluster as a whole. On the other hand, as suggested previously (Douris et al., 1995) and supported also from the reconstructed phylogeny (this study), it seems that the formation of the A. turrita clade has originally taken place in the western part of the Aegean region, presumably before the separation of this area from the Greek mainland. Extensive tectonic movements and volcanism in this area created an unstable coastline with several events of land submergence and reemergence (Anastassakis and Dermitzakis, 1990). These events, as well as other causes, may have brought about severe population bottlenecks. This hypothesis may explain the relatively small intraspeciwc diverentiation of A. turrita populations in the west Cyclades (Douris et al., 1995) compared to the neighboring A. caerulea populations. However, it is possible that small populations may have survived extensive sea-level changes at certain refugia ; the higher level of diverentiation of A. turrita on the island of Sifnos (evident also in Douris et al., 1995) suggests that such a refugium hypothesis could be employed for this island, and is supported also by data for other land snails (Mylonas, 1982). According to that hypothesis, older surviving populations of A. turrita that were initially restricted on Sifnos have later colonized the island of Kea and the volcanic island of Milos (but not other neighboring islands like Serifos or Kythnos) through relatively recent dispersal events. It seems that A. caerulea did not succeed to establish a viable population on the western part of the Cyclades. The major Wnding of our phylogenetic analysis, concerning the clade of A. caerulea and A. brevicollis, is the clear separation of group B a set of taxa with a southeast distribution from the rest of the taxa (the relevant node is indicated with an arrow at Fig. 3). This unambiguous relation of geography with phylogeny strongly suggests strongly that vicariance is the major factor that shaped the distribution of the members of the caerulea brevicollis clade. Yet, which palaeogeographic event was the cause of this separation attributed to vicariance? We have already mentioned that there are two candidate vicariance events; either the formation of the mid-aegean channel (12 9 MYA), or the permanent separation of the areas across this channel after the end of Messinian salinity crisis (6 4 MYA). Although a clocklike evolutionary rate is rejected by our analysis, in the estimation with the relaxed clock assumptions, the post salinity crisis calibration (at 6 4 MYA) resulted in better concordance between phylogenetic and palaeogeographic separations, whereas the alternative calibration (at 12 9 MYA) produced discrepancies (Table 3). We must notice however, that in deep nodes both calibrations produce severe discrepancies with palaeogeographic events as well as high variance, presumably due to insuycient sample of species and populations outside A. caerulea and A. brevicollis and/or the potentially high variance in rates due to a relatively short alignment length. The present geographic distribution patterns of groups A and B revealed by our analysis (Fig. 4) are generally explained through the vicariance hypothesis, no matter which calibration is selected. On the other hand, certain haplotypes of group A (Tilos, Kandeliousa, Kos) at the eastern part of the channel and one haplotype (Anm1 from AnaW) of group B at the west part cannot be readily attributed to any known vicariance event. A number of alternative explanations, which are not necessarily mutually exclusive, may be employed for these exceptions. One possibility is that haplotypes of group A found in the eastern part of the channel are remnants of ancestral polymorphism preserved within the area occupied now by group B. Based on this scenario, one would expect these southeast haplotypes to have a more basal position within clade A, but this is not the case (on the contrary, some are strikingly similar or even identical to typical group A haplotypes, for example bre.til1 with cae.amo1). Furthermore, ancestral polymorphisms persist less if evective population size is small and in populations that exhibit a stepping stone model (Edwards and Beerli, 2000) like Albinaria (Schilthuizen and Lombaerts, 1994; Giokas and Mylonas, 2004). A second alternative concerns post-vicariance dispersal events, involving either the formation of temporal land bridges connecting the respective areas or long distance dispersal over sea. A. caerulea is distributed in the Cyclades and on the coast of Turkey at the same latitude, whereas A. brevicollis occurs on the southeast Aegean and the neighboring coast of Turkey. Occasional land bridges that could have occurred in the area subsequent to the permanent formation of the mid-aegean trench may have been used as dispersal corridors for these haplotypes. A passive dispersal scenario, however, gets more support if we recall that Albinaria dwells on marble and limestone rocks, which were transported and used for building in ancient times (Waelkens et al., 1988; Tykot and Ramage, 2002) and that some of these haplotypes (bre.anm1 and 2 from AnaW and bre.kos2 from Kos) are found at archaeological sites (ancient temples) or at ports or very close to them. Such

11 1234 V. Douris et al. / Molecular Phylogenetics and Evolution 44 (2007) human-aided dispersal has been already suggested for Albinaria or relative clausiliid taxa such as Isabellaria (Welter- Schultes, 1998; Uit de Weerd et al., 2005). Other types of passive long distance dispersal, such as aerial dispersal by birds (Preece and Gittenberger, 2003; Gittenberger et al., 2006) or sea rafting (Mylonas, 1984; Douris et al., 1998a) have been proposed for snails and are probable, yet still hypothetic, for Albinaria. Another option, employing also some kind of dispersal, is to explain the presence of these haplotypes at the wrong side of the channel as the result of hybridization either at occasional contact zones or following some dispersal event; hybridization might have eliminated most of the introduced polymorphism in nuclear markers but the mtdna lineages could have persisted, given the uniparental mode of inheritance of mtdna. In order to fully investigate this option, a comparison with relevant phylogenies based on nuclear data is required. Some insight can be provided collectively from available literature: there is one study that used nuclear data (ITS) for inferring phylogenies in the caerulea brevicollis group (van Moorsel, 2001), including A. brevicollis from Kos and Turkey. The population from Kos clusters within A. caerulea (group A) taxa using both nuclear (ITS; van Moorsel, 2001) and mtdna (COII; Douris, 1997; 16S rrna-atpase8, this study) phylogenies, thus indicating that hybridization is not the case, at least for this taxon. However, the introgression hypothesis cannot be completely rejected, since hybridization among sympatric Albinaria species has been extensively reported (Kemperman and Degenaars, 1992; Schilthuizen, 1994; Giokas et al., 2000; van Moorsel, 2001). Furthermore, it seems that within the caerulea brevicollis group there are weak reproductive isolation barriers, especially pre-mating, between diverent populations (Giokas et al., 2006). A novel Wnding of this work is the identiwcation of three subgroups within group A, which exhibit a certain extent of geographic pattern (a1 North, a2+ Center, a3 South; Fig. 4). The separation of subgroup a3 from a1 and a2+ might correspond to the separation of the southern Cyclades islands from the northern Cyclades plateau that occurred 3.5 MYA (Fig. 2) and had a signiwcant impact on present day distribution of certain Aegean taxa (see Kasapidis et al., 2005). The subsequent separation between Center and North subgroups is probably more recent and is associated with the erratic fragmentation of this part of the Cyclades. This compartmentalization is accompanied by a radiation of A. caerulea, which seems stochastic and implies that populations of A. caerulea have undergone repeated bottleneck episodes, due to sea-level changes during the Pleistocene, and are subject to genetic drift or diverential selection. Correlation of A. caerulea contemporary distributions with events more recent than fragmentation of the Cyclades plateau, may employ investigation of population variability using diverent types of molecular markers and analysis tools. The impact of very recent historical events, such as the volcanic activity of Thira (expected to cause severe bottlenecks in the neighboring regions) or human-aided colonization of Vravrona (possibly subjected to genetic drift), might indicate possible stochastic processes that contribute in shaping present day distributions in addition to palaeogeography. Our Wndings are in concordance with hypotheses of recent radiations associated with the palaeogeographic history of the Aegean, such as those exhibited by the lizards Podarcis (Poulakakis et al., 2003, 2005) and Cyrtopodium kotschyi (Kasapidis et al., 2005), frogs (Beerli et al., 1996) and salamanders (Weisrock et al., 2001). Nevertheless, we must notice that among these taxa (including Albinaria) there are considerable diverences in the inferred sequence of evolutionary events (sometimes far from parsimonious). Those probably rexect intrinsic properties of each taxon and imply that the erratic palaeogeography of this region is not suycient to explain current diverentiation patterns. Such properties include dispersal abilities, population structure and reproductive isolation. Albinaria is much less capable of active dispersal compared to the above-mentioned vertebrate taxa and therefore land bridges are more essential for dispersal to occur. Furthermore, the evolutionary history of the caerulea brevicollis group divers from the one inferred for Mastus (Parmakelis et al., 2005), another land snail in that area. Parmakelis et al. (2005) have proposed that the radiation of Mastus corresponds better to older geological events (mid-aegean trench) and is more stochastic afterwards. However, Mastus divers from Albinaria in several aspects. Mastus is cryptic and forms less dense populations than Albinaria (Giokas et al., 2000; Giokas and Mylonas, 2004; Parmakelis and Mylonas, 2004), and interspeciwc reproductive isolation in Mastus, contrary to Albinaria, is complete and suggests an older taxon (Parmakelis et al., 2005). Thus, in Mastus there was suycient time for diverentiation, and introgression and dispersal are less likely. The diverence in the inferred evolutionary patterns between the two genera does not mean that the major distributional pattern was not shaped by vicariance, but rather implies that populations of Albinaria have responded partly stochastically to erratic historical events. This is not surprising for these taxa since they occupied this region before its fragmentation, and especially for these land snails that exhibit strong population structure and some level of hybridization. Acknowledgments This work was supported by the National and Kapodistrian University of Athens (ELKE Grants 70/4/4231, 70/4/ 7805, to G.C.R. and R.L.), by the Greek State Scholarships Foundation (post-doctoral fellowship to V.D.), and by PRAXIS XXI program of the Portuguese Ministry for Science and Technology (to D.T.). We thank M. Mihalatos for his technical assistance, A. Parmakelis for helping with the r8s analysis, and two anonymous reviewers for valuable comments and suggestions.