Abstract

The aims of the study were to infer phylogeographic relationships between the populations of the minute, dioecious, spring-inhabiting snail Grossuana from the Balkans, and to interpret the resulting pattern in the context of geological history of the region. The cytochrome c oxidase subunit I gene was sequenced from 23 previously unstudied populations of Grossuana from Bulgaria and analysed together with published sequences from the other populations of Grossuana from the Balkans. In Bulgaria, six clades or putative clades (lacking statistical support) were identified. Within the clades the p-distances were in the range 0.2–0.9% and between the clades 1.6–3.4%. Among all 33 studied populations, 42 haplotypes were found (haplotype diversity = 0.955; nucleotide diversity π = 0.059). All of the haplotypes from Bulgaria and Romania formed a clade that was distinct from all of the Serbian and Greek haplotypes. At the estimated divergence time of 3.60 ± 0.58 ma a sea connection between the Pannonian Sea and Aegean Sea (at the site of the present Velika Morava Valley) formed a dispersal barrier for these freshwater snails. The nucleotide diversity within the Bulgarian/Romanian lineage was lower (π = 0.019, 41 polymorphic sites) that within the Serbian/Greek group (π = 0.049, 70 polymorphic sites), perhaps as a result of bottlenecks during the Pleistocene glaciations. Within the Bulgarian populations, all of the diversity originated in the Pleistocene, during the Calabrian (estimated time 1.26–1.42 ma). During the Pleistocene, the unstable system of rivers and lakes in southwestern Bulgaria, with glaciers in the Pirin and Rila Mountains, probably resulted in the extinction of Grossuana in SW Bulgaria. Subsequently, this territory was likely recolonized from eastern Bulgarian populations.

INTRODUCTION

The rich fauna of the Balkans, including hydrobioid gastropods, has been considered as a product of the complicated geological history of the region, which has served as a refugium during glaciations and been subjected to numerous sea-level fluctuations (Creutzburg, 1963; Kougioumoutzis, Simaiakis & Tiniakou, 2014). Against this background we have studied phylogeographic diversification of the minute, hydrobioid spring snail Grossuana and here attempt to interpret the observed pattern in the context of the complex geological history of the region.

The freshwater truncatelloideans may have appeared in the Early Carboniferous (Kabat & Hershler, 1993) and they are therefore suitable models for the evaluation of old (pre-Pleistocene) biogeographic relationships. The distributions of freshwater Truncatelloidea have been interpreted in terms of Neogene drainage patterns and subsequent fragmentation by changes in climate and landscape. In particular, spring snails have been discussed in this way (Falniowski & Szarowska, 2011a), as assuming that the springs inhabited by those obligatorily aquatic animals are stable habitats and that gene flow among the springs is very low. There have been many studies on the phylogeny, population genetic structure and gene flow of the spring fauna, including gastropods (e.g. Colgan & Ponder, 1994; Ponder, Eggler & Colgan, 1995; Bilton, Freeland & Okamura, 2001; Finston & Johnson, 2004; Hershler & Liu, 2004a, b; Falniowski, Szarowska & Sirbu, 2009; Osikowski et al., 2015). Most of the studies point to low levels of gene flow and high levels of endemism in spring snails (e.g. Colgan & Ponder, 1994; Ponder et al., 1995; Finston & Johnson, 2004), although some of the species are rather widespread, with much gene flow among their populations (Falniowski et al., 1998; Falniowski, Mazan & Szarowska, 1999; Hershler, Mulvey & Liu, 2005).

The truncatelloidean fauna of the Balkan Peninsula includes many taxa with a simplified shell lacking characteristic traits, a penis with a more or less prominent double or single lobe on the left edge and female reproductive organs with a loop of the oviduct, bursa copulatrix and two seminal receptacles. Such morphological characters have long been used for the descriptions of new species belonging to this group. However, molecular studies have revealed that anatomical traits do not provide sufficient evidence to establish phylogenetic relationships between higher taxa or to distinguish closely related species (Falniowski et al., 2012).

Grossu (1946) described a new species of minute hydrobioid gastropod, Paladilhiopsis codreanui, from a spring at Techirghiol Lake in Romania, although first collected near Balčic in Bulgaria (Grossu, 1986). Radoman (1966) described a new species of Pseudamnicola vurliana from Kamena Vurla in Greece. Subsequently, Radoman (1973) described a new genus, Grossuana, for which the type species was the newly described G. serbica. He assigned both P. codreanui and P. vurliana to the genus Grossuana and described three additional new species of the genus, which were later (Radoman, 1983) considered to be subspecies of G. serbica. Grossuana ranges from Serbia through Macedonia to northern Greece, Bulgaria and southeastern Romania (Radoman, 1985) (Fig. 1). Szarowska et al. (2007) added two Greek species to this genus, G. haesitans (Westerlund, 1881) from the spring of the Louros River and G. delphica (Radoman, 1973) from the spring at Delphi. In a broader study, Falniowski et al. (2012) found that the distribution of Grossuana is disjunct, with part of the distribution covering areas of Serbia, Bulgaria and Romania, while another covers northeastern Greece. The latter includes the spring of Achilles northwest of Lamia, two springs on the Volos peninsula (Pilion Mt.) and one on Evvoia Island [these three identified as G. marginata (Westerlund, 1881)] and the spring of Athena in the Thembi Valley [G. hohenackeri (Küster, 1853)] (Fig. 1). Falniowski et al. (2012) demonstrated that using morphological data alone (the shell as well as the reproductive organs) it was not possible either to determine or distinguish species or to separate representatives of Grossuana and RadomaniolaSzarowska, 2006, despite the fact that the genera are not sister taxa (Szarowska, 2006).

Figure 1.

Map of all localities sampled in phylogenetic analyses. Localities B1–B23 are represented by new COI sequences; localities A–J represented by published sequences (Szarowska et al., 2007; Falniowski et al., 2012). Hatched areas: Grossuana range (after Radoman, 1985). Geographical features discussed in the text are indicated. See Table 1 for locality details.

Figure 1.

Map of all localities sampled in phylogenetic analyses. Localities B1–B23 are represented by new COI sequences; localities A–J represented by published sequences (Szarowska et al., 2007; Falniowski et al., 2012). Hatched areas: Grossuana range (after Radoman, 1985). Geographical features discussed in the text are indicated. See Table 1 for locality details.

Additional species of Grossuana have been described more recently. Considering shell characters as well as the penis, Glöer & Georgiev (2009) described G. angeltsekoviGlöer & Georgiev, 2009 (from springs in the West Rhodopes Mts and the lower slopes of the Pirin Mts in the Mesta River Valley) and G. thracicaGlöer & Georgiev, 2009 (from Chirpan Bunar spring, in the Upper Thracian Lowland, southern Bulgaria). Georgiev (2012) described G. aytosensisGeorgiev, 2012 (from a water source near Aytos, eastern Stara Planina Mts) and G. radostinaeGeorgiev, 2012 (from a stream near Madara, northeastern Bulgaria). Georgiev & Glöer (2013) described two additional species, G. slavyanicaGeorgiev & Glöer, 2013 (from Slavyanka Mts., southwest Bulgaria) and G. derventicaGeorgiev & Glöer, 2013 (from Dervent Heights, southeastern Bulgaria) and Georgiev et al. (2015) described G. falniowskiiGeorgiev et al., 2015 (from spring of the Bedechka River, Krayrechen Park, Stara Zagora, central Bulgaria).

The aim of this study was to infer, using the mitochondrial cytochrome c oxidase gene (COI) as a molecular marker, phylogeographic relationships between all the populations of Grossuana studied so far and to interpret the resulting pattern in the context of the geological history of the region. It was not our intention to evaluate the validity of species-level taxa in this genus.

MATERIAL AND METHODS

Sample collection and fixation

One of us (D.G.) collected Grossuana snails from 23 localities from throughout Bulgaria (Fig. 1, Table 1) and determined them by means of morphological characters and their localities. The samples included representatives of six Grossuana species recently described by D.G. (G. angeltsekovi, G. aytosensis, G. codreanui, G. falniowskii, G. radostinae and G. slavyanica, mostly paratypes) and one Radomaniola species (R. bulgarica). The snails were collected by hand or with a sieve. Individuals to be used for molecular analyses were washed in 80% ethanol, in which they were left to stand for c. 12 h. The ethanol was subsequently changed twice over 48 h and finally transferred to 96% ethanol after a few days. Samples were stored at −20 °C prior to DNA extraction. Shells were photographed under a Nikon SMZ18 stereomicroscope with dark field illumination using a Canon EOS 50D digital camera.

Table 1.

Sampling localities of Grossuana with their geographical coordinates and the haplotypes of the COI gene detected at each locality.

Locality ‘taxon’ Site Coordinates
 
COI haplotypes 
B1  Kavarna town, Bulgaria 43°24′47″N 28°21′04″E HB1A × 2, HB1B 
B2  Kovach spring, Krepcha village, Bulgaria 43°26′58″N 26°06′51″E HB2A, HB2B × 2 
B3 Grossuana codreanui Balchik town, Bulgaria 43°13′37″N 28°00′08″E HB1A × 4 
B4  Aladzha monastery, Bulgaria 43°16′39″N 28°01′04″E HB1A × 3 
B5 Grossuana radostinae Madara village, Bulgaria 43°09′36″N 27°03′36″E HB1A 
B6  Tserovo village, Bulgaria 43°00′25″N 23°20′33″E HB6 × 2 
B7  Iskrec village, Bulgaria 42°59′46″N 23°14′00″E HB6 × 9 
B8 Grossuana aytosensis No. of Aytos town, Bulgaria 42°42′52″N 27°16′09″E HB8A, HB8B, HB8C × 3 
B9  Pekeyuka, karst spring, Bulgaria 42°47′16″N 22°59′26″E HB6 × 2 
B10  Bosnek village, water source, Bulgaria 42°30′11″N 23°10′57″E HB10A × 2, HB10B 
B11  Bosnek village, Popov izvor spring, Bulgaria 42°30′22″N 23°10′36″E HB10A, HB11 × 2 
B12 Radomaniola bulgarica Ostra Mogila village, Bulgaria 42°27′11″N 25°28′27″E HB12 × 2 
B13 Grossuana falniowskii Stara Zagora city, spring, Bulgaria 42°26′52″N 25°38′03″E HB13 × 6 
B14  Smolichane village, Bulgaria 42°07′58″N 22°48′25″E HB11 × 2 
B15  Vaksovo village, spring, Bulgaria 42°09′23″N 22°51′28″E HB11 × 2 
B16 Grossuana angeltsekovi Belashtitza village, spring, Bulgaria 42°03′13″N 24°44′10″E HB16A × 2, HB16B, HB16C × 4 
B17  Krichim town, Bulgaria 42°01′22″N 24°28′08″E HB17 × 2 
B18  Bachkovo town, water source, Bulgaria 41°56′08″N 24°51′46″E HB16C × 2 
B19  Vodnata dranchi dupka, Bulgaria 42°02′55″N 26°32′15″E HB19A × 3, HB19B, HB19C 
B20  Vitanovo spring, Bulgaria 42°00′39″N 27°25′03″E HB20 × 5 
B21  Gotse Deltchev town, karst spring, Bulgaria 41°35′00″N 23°41′37″E HB21A, HB21B, HB21C, HB21D, HB21E 
B22  Petrovo village, karst spring, Bulgaria 41°25′42″N 23°31′34″E HB22A, HB22B 
B23 Grossuana slavyanica Goleshovo village, Bulgaria 41°25′53″N 23°35′19″E HB22B 
Grossuana codreanui Jasenovo, Bulgaria (EF061920)   HB 
Grossuana codreanui Techirghiol Lake, Romania (EF061919) 43°59′37″N 28°32′46″E HB1A 
Grossuana serbica Serbia - Raška river Spring—(EF061921) 43°06′57″N 20°22′15″E HS 
Grossuana marginata Spring between Loutsa and Steni, Evvoia island, Greece (KC011765) 38°35′16″N 23°48′57″E HG1 
Grossuana delphica Kastalia spring, Delphi, Greece—(EF061922) 38°28′59″N 22°30′19″E HG2 
Grossuana hohenackeri Spring of Athena, Tembi Valley, Greece (KC011748–KC011750) 39°58′26″N 22°38′17″E HG3, HG4, HG5 
Grossuana vurliana Spring of Louros River, Greece (EF061923) 39°25′56″N 20°50′30″E HG6 
Grossuana sp. Spring of Achilles, ESE of Kalamakion, Greece (KC011746) 38°59′13″N 22°22′43″E HG7 
Grossuana sp. Spring E of Anilion, Oros Pilion, Greece (KC011767–KC011769) 39°24′49″N 23°09′23″E HG8, HG9, HG10 
Grossuana sp. Spring NW of Dhrakia, Oros Pilion, Greece (KC011770–KC011771) 39°23′36″N 23°02′33″E HG11, HG12 
Locality ‘taxon’ Site Coordinates
 
COI haplotypes 
B1  Kavarna town, Bulgaria 43°24′47″N 28°21′04″E HB1A × 2, HB1B 
B2  Kovach spring, Krepcha village, Bulgaria 43°26′58″N 26°06′51″E HB2A, HB2B × 2 
B3 Grossuana codreanui Balchik town, Bulgaria 43°13′37″N 28°00′08″E HB1A × 4 
B4  Aladzha monastery, Bulgaria 43°16′39″N 28°01′04″E HB1A × 3 
B5 Grossuana radostinae Madara village, Bulgaria 43°09′36″N 27°03′36″E HB1A 
B6  Tserovo village, Bulgaria 43°00′25″N 23°20′33″E HB6 × 2 
B7  Iskrec village, Bulgaria 42°59′46″N 23°14′00″E HB6 × 9 
B8 Grossuana aytosensis No. of Aytos town, Bulgaria 42°42′52″N 27°16′09″E HB8A, HB8B, HB8C × 3 
B9  Pekeyuka, karst spring, Bulgaria 42°47′16″N 22°59′26″E HB6 × 2 
B10  Bosnek village, water source, Bulgaria 42°30′11″N 23°10′57″E HB10A × 2, HB10B 
B11  Bosnek village, Popov izvor spring, Bulgaria 42°30′22″N 23°10′36″E HB10A, HB11 × 2 
B12 Radomaniola bulgarica Ostra Mogila village, Bulgaria 42°27′11″N 25°28′27″E HB12 × 2 
B13 Grossuana falniowskii Stara Zagora city, spring, Bulgaria 42°26′52″N 25°38′03″E HB13 × 6 
B14  Smolichane village, Bulgaria 42°07′58″N 22°48′25″E HB11 × 2 
B15  Vaksovo village, spring, Bulgaria 42°09′23″N 22°51′28″E HB11 × 2 
B16 Grossuana angeltsekovi Belashtitza village, spring, Bulgaria 42°03′13″N 24°44′10″E HB16A × 2, HB16B, HB16C × 4 
B17  Krichim town, Bulgaria 42°01′22″N 24°28′08″E HB17 × 2 
B18  Bachkovo town, water source, Bulgaria 41°56′08″N 24°51′46″E HB16C × 2 
B19  Vodnata dranchi dupka, Bulgaria 42°02′55″N 26°32′15″E HB19A × 3, HB19B, HB19C 
B20  Vitanovo spring, Bulgaria 42°00′39″N 27°25′03″E HB20 × 5 
B21  Gotse Deltchev town, karst spring, Bulgaria 41°35′00″N 23°41′37″E HB21A, HB21B, HB21C, HB21D, HB21E 
B22  Petrovo village, karst spring, Bulgaria 41°25′42″N 23°31′34″E HB22A, HB22B 
B23 Grossuana slavyanica Goleshovo village, Bulgaria 41°25′53″N 23°35′19″E HB22B 
Grossuana codreanui Jasenovo, Bulgaria (EF061920)   HB 
Grossuana codreanui Techirghiol Lake, Romania (EF061919) 43°59′37″N 28°32′46″E HB1A 
Grossuana serbica Serbia - Raška river Spring—(EF061921) 43°06′57″N 20°22′15″E HS 
Grossuana marginata Spring between Loutsa and Steni, Evvoia island, Greece (KC011765) 38°35′16″N 23°48′57″E HG1 
Grossuana delphica Kastalia spring, Delphi, Greece—(EF061922) 38°28′59″N 22°30′19″E HG2 
Grossuana hohenackeri Spring of Athena, Tembi Valley, Greece (KC011748–KC011750) 39°58′26″N 22°38′17″E HG3, HG4, HG5 
Grossuana vurliana Spring of Louros River, Greece (EF061923) 39°25′56″N 20°50′30″E HG6 
Grossuana sp. Spring of Achilles, ESE of Kalamakion, Greece (KC011746) 38°59′13″N 22°22′43″E HG7 
Grossuana sp. Spring E of Anilion, Oros Pilion, Greece (KC011767–KC011769) 39°24′49″N 23°09′23″E HG8, HG9, HG10 
Grossuana sp. Spring NW of Dhrakia, Oros Pilion, Greece (KC011770–KC011771) 39°23′36″N 23°02′33″E HG11, HG12 

Sequences from GenBank are also included (labelled A–J, with GenBank numbers; Szarowska et al., 2007; Falniowski et al., 2012).

DNA extraction and sequencing

DNA was extracted from foot tissue using a Sherlock extraction kit (A&A Biotechnology) and dissolved in 20 µl of tris-EDTA buffer. Polymerase chain reaction (PCR) was performed in a reaction mixture with a total volume of 50 µl using the primers LCOI490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) (Folmer et al., 1994) and COR722b (5′-TAAACTTCAGGGTGACCAAAAAATYA-3′) (Wilke & Davis, 2000) for COI. The PCR conditions were as follows: an initial denaturation step of 4 min at 94 °C, followed by 35 cycles at 94 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min, with a final extension of 4 min at 72 °C. A 10 µl sample of the PCR product was run on a 1% agarose gel to check the quality of the PCR product. The PCR product was purified using Clean-Up columns (A&A Biotechnology). The purified PCR product was then sequenced in both directions using BigDye Terminator v. 3.1 (Applied Biosystems), following the manufacturer's protocol and using the primers indicated above. The products of the sequencing reaction were purified using ExTerminator Columns (A&A Biotechnology), and the sequences were read using an ABI Prism sequencer.

Data analysis

Sequences were aligned and edited in Bioedit v. 7.1.3.0 (Hall, 1999). Basic sequence statistics, including haplotype polymorphism and nucleotide divergence, were calculated in DnaSP v. 5.10 (Librado & Rozas, 2009). The saturation test was performed using DAMBE (Xia, 2013).

In a phylogenetic analysis, 15 other sequences from GenBank were used as a reference (Table 1) and Daphniola exigua (GenBank JF916470; Falniowski & Szarowska, 2011b), the type species of the genus phylogenetically closest to Grossuana, was used as the outgroup. The data were analysed using approaches based on Bayesian inference and maximum likelihood (ML). We applied the GTR + I + Γ model, which is the only nucleotide substitution model implemented in RaxML (Stamatakis, 2014).

The Bayesian analyses were run using MrBayes v. 3.2.3 (Ronquist et al., 2012) with the default priors. Two simultaneous analyses were performed, each of which lasted 10,000,000 generations, with one cold chain and three heated chains, starting from random trees and sampling the trees every 1,000 generations. The first 25% of trees were discarded as burn-in. The analyses were summarized as a 50% majority-rule tree.

A ML approach was applied in RAxML v. 8.0.24. One thousand searches were initiated with starting trees obtained through the randomized stepwise addition maximum parsimony method. The tree with the highest likelihood score was considered as the best representation of the phylogeny. Bootstrap support was calculated with 1,000 replicates and summarized on the best ML tree. RAxML analyses were performed using the free computational resource CIPRES Science Gateway (Miller, Pfeiffer & Schwartz, 2010).

Median-joining calculations, as implemented in NETWORK v. 4.6.1.1 (Bandelt, Forster & Röhl, 1999), was used to infer the COI haplotype network. To test the validity of the molecular clock assumption, the likelihoods for trees with and without the molecular clock were calculated with PAUP in a likelihood ratio test (LRT) (Nei & Kumar, 2000). The relative rate test (RRT) (Tajima, 1993) was performed in MEGA6 (Tamura et al., 2013). As Tajima's RRTs and the LRT test rejected an equal evolutionary rate throughout the tree for Grossuana, time estimates were calculated using a penalized-likelihood method (Sanderson, 2002) in r8s v. 1.7 for Linux (Sanderson, 2003). To calibrate the molecular clock, two hydrobiids, Peringia ulvae (AF478401) and Salenthydrobia ferreri (AF478410) were used as outgroups. The divergence time between those two species (proposed by Wilke, 2003, with correction by Falniowski et al., 2008), callibrated the nodes within Grossuana.

RESULTS

In total we obtained 79 COI sequences of length 552 bp (GenBank Accession numbers KU201035–KU201113). No saturation was revealed by the test of Xia et al. (2003). Among all of the Grossuana COI sequences analysed, 42 haplotypes (haplotype diversity h = 0.955; nucleotide diversity π = 0.059) were identified. The Bulgarian haplotypes (including one sequence from Romania) formed a lineage that was clearly separated from the Serbian/Greek Grossuana (bootstrap probability = 70%; Bayesian posterior probability = 0.89), with an estimated separation time of 3.60 ± 0.58 ma (Clades I–VI; Fig. 2). The nucleotide diversity within the Bulgarian Grossuana lineage was much lower (π = 0.019, 41 polymorphic sites) in comparison with that within the Serbian/Greek clade (π = 0.049, 70 polymorphic sites).

Figure 2.

A. Maximum-likelihood phylogram of Grossuana COI haplotypes. Haplotypes sequenced in present work are indicated in bold; previously published sequences are shown in plain text. See Table 1 for localities of haplotypes. Bootstrap support and Bayesian posterior probabilities are shown (when greater than 50% or 0.5, respectively). B. Median joining tree for Bulgarian COI haplotypes.

Figure 2.

A. Maximum-likelihood phylogram of Grossuana COI haplotypes. Haplotypes sequenced in present work are indicated in bold; previously published sequences are shown in plain text. See Table 1 for localities of haplotypes. Bootstrap support and Bayesian posterior probabilities are shown (when greater than 50% or 0.5, respectively). B. Median joining tree for Bulgarian COI haplotypes.

The Bulgarian Grossuana haplotypes formed six main clades (Clades I–VI; Figs 2A, B, 3), some lacking statistical support, between which p-distances ranged from 1.3 to 3.4% (Table 2). This corresponds to an estimated divergence time between the six Bulgarian clades of 1.26–1.42 Mya. The p-distances within the clades were small, ranging from 0.2 to 0.9%. Clade I from southwestern Bulgaria was characterized by14 haplotypes, but most of them differed by only one substitution from the closest haplotype (Figs 2B, 3). Within Clade I, the most divergent haplotypes correspond to G. slavyanica (Fig. 2A). Putative Clades II and III, from southeastern Bulgaria (Fig. 3), were unsupported and their phylogenetic relationships were not resolved (Figs 2, 3); they may represent one clade, although the haplotype network (Fig. 2B) suggests their distinctness. Haplotypes belonging to both of these putative clades were found at one of the localities (B8, including a paratype of G. aytosensis). Putative Clade III also included haplotypes from localities B19 and B20.

Table 2.

COI p-distances between the main Grossuana clades and putative clades (I–VI).

 II III IV VI 
0.009      
II 0.019 0.002     
III 0.025 0.013 0.006    
IV 0.026 0.023 0.018 0.004   
0.029 0.018 0.016 0.017 0.007  
VI 0.034 0.016 0.028 0.024 0.018 0.006 
 II III IV VI 
0.009      
II 0.019 0.002     
III 0.025 0.013 0.006    
IV 0.026 0.023 0.018 0.004   
0.029 0.018 0.016 0.017 0.007  
VI 0.034 0.016 0.028 0.024 0.018 0.006 

Italics indicate within-group genetic differentiation.

Figure 3.

Geographical distribution of Bulgarian COI clades. Colour scheme of clades as in Figure 2. See Table 1 for locality details.

Figure 3.

Geographical distribution of Bulgarian COI clades. Colour scheme of clades as in Figure 2. See Table 1 for locality details.

Clades IV and V (the latter unsupported; Fig. 2A) were each represented at a single locality: Clade IV from northern Bulgaria (locality B2) and putative Clade V from central Bulgaria (locality B13) (Fig. 3). In addition to population B13 (described as G. falniowskii), putative Clade V also included a published Bulgarian sequence (HB haplotype, assigned to G. codreanui by Szarowska et al., 2007); the p-distance between these two haplotypes was 0.7%.

Haplotypes from locality B12 (described as Radomaniola bulgarica), situated close to B13, were assigned to Clade VI (Figs 2A, 3). The remaining haplotypes forming this clade came from northeastern Bulgaria. The p-distance between haplotype B12 and haplotypes from northeastern populations was 0.008. Published sequences from Romania were also assigned to Clade VI. Our samples identified as G. codreanui and G. radostinae carried the same haplotype in Clade VI.

The shells of Grossuana belonging to the molecularly inferred clades and putative clades are shown in Supplementary Material Figures S1 and S2. The shells of Clade I (Supplementary Material Fig. S1), represented by 10 populations, are illustrated for 2 populations: B11 (Supplementary Material Fig. S1A–H) and B9 (Supplementary Material Fig. S1I–L). Wide variability of the shell proportions, especially spire height, was observed in population B11, while in population B9 all of the shells were low-spired with a broad aperture. However, these two populations of Clade I encompass all of the shell variability observed in the Bulgarian Grossuana (cf. Supplementary Material Figs S1, S2). The shells of the other clades (Supplementary Material Fig. S2), found in one to four populations each, showed less variation within each clade. In Clade II (population B8: Supplementary Material Fig. S2A–D) the shells were broad, with moderately high spires, and the shells of Clade III (population B19: Supplementary Material Fig. S2E, F) were similar. In Clade IV (population B2: Supplementary Material Fig. S2G–I) the shells were relatively high-spired, whereas in Clade V (population B13: Supplementary Material Fig. S2J–N) the spire was low and the mouth broad. In Clade VI (population B3: Supplementary Material Fig. S2O–Q), the mouth was less broad, but the spire was high or low.

DISCUSSION

Like other members of the rich European fauna of minute snails belonging to the superfamily Truncatelloidea, the genus Grossuana was long described only with reference to its morphological characters (e.g. Grossu, 1946, 1986; Radoman, 1973, 1983, 1985; Glöer & Georgiev, 2009; Georgiev, 2012, 2013; Georgiev et al., 2015). However, the application of molecular data in taxonomic and phylogeographic studies of Grossuana (Szarowska et al., 2007; Falniowski et al., 2012) has shown that when the morphology of the shell and the reproductive organs is used alone, it is impossible to achieve even species determination within this group. Thus, molecular markers must be applied to distinguish the true biological entities.

Our molecular results distinguished a major clade of Grossuana in Bulgaria and Romania, which was clearly separated from the other taxa of the genus known from Serbia and Greece. Similar distinctness of the Bulgarian vs Dinaric haplotypes have been found in the western capercaillie (Bajc et al., 2011), a turtle (Fritz et al., 2006) the nose-horned viper (Ursenbacher et al., 2008) and in newts (Wielstra et al., 2013).

The modern post-Alpine topography of the Mediterranean emerged in the late Tortonian (8 ma) (Kostopoulos, 2009). According to Popescu et al. (2009) and Suc et al. (2011), the Balkan region suffered drastic environmental changes during the Messinian salinity crisis. During the Miocene and Pliocene, a shallow sea, the Pannonian Basin, filled the part of Central Europe currently known as the Pannonian Plain. The Pannonian Basin was connected through the Iron Gates with the Dacic Basin, a vast water body that filled the area between the Carpathians and the Balkan mountains (Popov et al., 2004, 2006; Clauzon et al., 2005; Popescu et al., 2009). As a part of the Paratethys system, the Pannonian and Dacic Basins were connected with the Euxinian Basin to the east and directly with the present Aegean Sea to the south. This saltwater connection, known as the Balkan Gateway, existed through the graben valley of the present Velika (Big) and Južna (South) Morava rivers, which was formed during the Neogene as a result of tectonic movements (Stoyanov & Gachev, 2012). In the Miocene, Pliocene and lower Pleistocene, this region was submerged by the Pannonian Sea. Finally, the present Velika Morava Valley was cut into the floor of a former bay of the Pannonian Sea. The presence of a saltwater connection between the Pannonian Basin and the Aegean Sea (5.60–1.8 ma) and, more recently, a wide river valley, may explain the distinctness of the Serbian/Greek populations of Grossuana from the Bulgarian/Romanian populations. Considering the present relatively restricted geographical range of Grossuana in comparison with Bythinella or Pseudamnicola, for example, the dispersal potential of Grossuana appears to be low. Thus, the sea that existed in the past and even the broad river valley with no calcium-rich springs formed an effective barrier. The isolating role of graben valleys was strengthened by the fact that glaciers were moving through these landforms during certain intervals (Stoyanov & Gachev, 2012), which presumably made them uninhabitable for freshwater snails.

According to a relaxed molecular clock, the separation of the Bulgarian/Romanian populations from the Serbian/Greek populations took place 3.6 ± 0.58 ma. This estimated time of divergence coincides with the existence of the connection between the Paratethys and the Aegean Sea described above, which likely formed a dispersal barrier for Grossuana. Our estimate was computed from the 5.9% p-distance between the two groups and using the rate 1.83 ± 0.21% per myr for COI provided by Wilke (2003). A similar estimate, of 1.62% per myr, was obtained by Hershler & Liu (2008), which also places the divergence time during the existence of the Paratethys/Aegean connection. Following this isolation, the Bulgarian/Romanian and Greek/Serbian groups evolved independently. The evidently lower diversity within the Bulgarian/Romanian group may reflect bottlenecks during the Pleistocene glaciations, as the glacial conditions in this northern region were presumably more severe than in Greece, which is situated further to the south.

Based on the topology of the COI tree and the geographical distribution of the Bulgarian/Romanian clades, we suggest that three main Grossuana groups can be distinguished: the Rhodopean (Clade I), Strandzhan (Clades II and III) and Balkan (Clades IV–VI) groups. Only the first of these is monophyletic in the COI analyses (Fig. 2) and the phylogenetic relationships between them are not resolved in our analyses. We speculate that the explanation for the separation of the Rhodopean and Strandzhan groups could be the ecological barrier formed by the Maritsa River Valley, a smaller-scale analogue of the Velika and Južna Morava Valleys discussed above.

The estimated time of divergence between the six Bulgarian/Romanian clades is 1.26–1.42 ma, corresponding to the Calabrian during the Pleistocene. In the Pleistocene, the unstable fluviolacustrine system in southwestern Bulgaria, with glaciers present in the Pirin and Rila Mountains (Zagorchev, 2007), probably formed effective, temporary barriers for Grossuana and caused its extinction in a large area of southwestern Bulgaria. Based on the available data, the small genetic differences among the Bulgarian populations of Clade I reflect the short history of Grossuana in the area, which was subsequently recolonized from elsewhere, perhaps from regions presently inhabited by Clades II–VI (this hypothesis could be tested by a more well-resolved phylogeny). In Clade I, the presence of relatively high haplotype diversity, coupled with low nucleotide diversity, agrees with the model of rapid population growth from an ancestral population with a small evolutionarily effective size (Avise, 2000). There must have been sufficient time for the recovery of haplotype variation via mutations, yet not enough time for the accumulation of larger sequence differences. This general interpretation appears to be appropriate for these spring-dwelling gastropods, whose populations may be established by a few immigrants, passively transported by birds or as passengers on windblown leaves.

It must be emphasized that species delimitation was not an objective of this study, for which larger samples, independent genetic markers and morphological data would be desirable. The study did not yield any strong evidence for the occurrence of more than one species at any one spring. While the presence of more than one truncatelloid species in one spring is not common, it has been noted for Bythinella (Radoman, 1976; Falniowski et al., 2009; Falniowski & Szarowska, 2011a), including Bulgarian species (Osikowski et al., 2015). We note that at locality B8 two haplotypes were recorded with a divergence of 1.3%, which is close to the interspecific threshold for COI of 1.5% suggested for Bythinella (Bichain et al., 2007). Georgiev (2012) described specimens from this locality as G. aytosensis, based on morphological characters, and the presence of such divergent haplotypes requires further study.

SUPPLEMENTARY MATERIAL

Supplementary material is available at Journal of Molluscan Studies online.

Acknowledgements

The study was supported by a grant from the National Science Centre (2012/05/B/NZ8/00407) to Magdalena Szarowska. We would like also to thank Associate Editor Robert Hershler, Editor David G. Reid and two anonymous reviewers for their valuable comments and suggestions.

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