Pseudamnicola Paulucci, 1878 (Caenogastropoda: Truncatelloidea) from the Aegean Islands: a long or short story?

Molecular phylogeny and putative species delimitation

The freshwater truncatelloideans in the Balkans appeared in the Palaeogene period, if not earlier (Kabat and Hershler 1993). Therefore, they are suitable for the evaluation of old (pre-Pleistocene) biogeographic relationships. Many of the present-day representatives of the freshwater Rissooidea are considered relicts, the distributions of which trace the Neogene drainage patterns fragmented by subsequent changes in climate and landscape (Hershler and Liu 2004). This hypothesis involves two conditions: (i) the springs inhabited by those obligatorily aquatic animals are stable habitats; and (ii) the gene flow among them, if at all present, is very low. In the abundant literature on the phylogeny and population genetic structure and gene flow of spring fauna, gastropods are well represented (e.g. Colgan and Ponder 1994; Ponder et al. 1995; Falniowski et al. 1998, 1999, 2009; Bohonak 1999; Hershler and Liu 2004; Brändle et al. 2005; Hershler et al. 2005; Falniowski and Szarowska 2011). Most studies indicate low levels of gene flow and high levels of endemism in spring snails (e.g. Colgan and Ponder 1994; Ponder et al. 1995; Finston and Johnson 2004), but other studies showed that some species were rather widespread, with high levels of gene flow among their populations (Falniowski et al. 1998, 1999; Hershler et al. 2005).

Phylogenetic analysis of Pseudamnicola indicates three main conclusions: (i) the studied populations of Pseudamnicola form 16 clades, according to the GMYC and ABGD results probably of species rank; (ii) intrapopulation differentiation level is generally low, slightly larger in populations occupying the mainland and islands close to the continent; and (iii) divergence times for most clades are less than 3 Mya. The short time of divergence, inferred from the low mitochondrial differentiation, suggesting thus close relationships between the studied Pseudamnicola populations, is confirmed by the low or no differentiation in the three studied nuclear loci.

According to the phylogenetic analysis and p-distance values for COI, the studied Pseudamnicola populations can be assigned to 16 clades, most of which are probably distinct species, according to the GMYC and ABGD analysis. The GMYC procedure has been applied in several previous studies (Barraclough et al. 2009; Papadopoulou et al. 2009a, b; Adolfsson et al. 2010; Pagès et al. 2010; Powell et al. 2011; Vuataz et al. 2011), including some on molluscs (Nekola et al. 2009; Lorion et al. 2010; Falniowski and Szarowska 2012; Jörger et al. 2012; Jörger and Schrödl 2013). Reliability of species delimitated by GMYC has also been discussed in the literature (e.g. Lohse 2009; Papadopoulou et al. 2009b; Jörger et al. 2012; Jörger and Schrödl 2013; Sauer and Hausdorf 2012), also with substantial criticism, and the method was further developed (Monaghan et al. 2009). However, in our work, the GMYC results were confirmed by ABGD analysis. Surely, with one short DNA fragment, species level distinction is always ambiguous; thus, we treat those clades as presumed species only, not attempting to distinguish and describe them formally—the latter should wait for more data available.

According to morphological criteria, six species of Pseudamnicola were distinguished in Greece (Schütt 1980; Szarowska et al. 2006). P. brachia was described from Crete (corresponding with clade C), P. chia from Khios (clade CH) and P. negropontina from Evvoia Island and may probably inhabit continental Greece as well (clade PN). P. pieperi is known from Karpathos (clade KAR). According to our results, P. pieperi may occur also on Rhodes Island; however, clades belonging to this cluster are more divergent, which rather suggests the existence of three rather than one species on these two islands. Recently, two molecularly distinct species from Rhodos were described (Radea et al. 2015). Similarly, P. negropontina may occupy the islands of Tinos, Naxos and probably Andros, but GMYC results indicate that there are four species in this area, with one case of sympatry (at locality A03). Two Pseudamnicola species come from the continent (Schütt 1980). P. macrostoma inhabits the northern Peloponnese and continental Greece. Unfortunately, we do not have well-fixed specimens of Pseudamnicola from this region, apart from the reference sequence (Radea et al. 2013). The second species, according to Schütt (1980), within the range of P. exilis (drawn of the map but not described and far from the type locality—see above) has been studied in the southern part of the Peloponnese and Kythira Island (Szarowska and Falniowski 2011). However, this presumed ‘P. exilis’ represented at least four distinct species, including one from Kythira Island, which was highly polymorphic (Fig. 29 in Szarowska and Falniowski 2011).

Morphological differentiation

Differences were noted in the shell habitus (height/width ratio, spire height, shell size) between the two presumed species of Pseudamnicola from the Peloponnese (population P01 vs P02 and P07) and two from the two localities in Kythira Island (Szarowska and Falniowski 2011: Figs. 2–14). No differences—apart from size—were found in the morphology of the penis and female reproductive organs between those populations (Szarowska and Falniowski 2011: Figs. 15–25 and 26); the lack of taxonomically useful characters and variable degrees of fixation-caused contraction may be an explanation for this.

On Andros Island, where there were three presumed species, shells were slightly variable and intrapopulation variability was sometimes higher than interpopulation variability. At locality A03, inhabited sympatrically by two presumed species, shell variation did not coincide with genotypic differences. Only the shells from localities A05 (not presented) and A06 were distinguishable, with their high penultimate whorls, from those of the other populations/species. However, PCA showed distinctness of the two Andros clades. The difference between the shells from the Naxos populations, N01 and N03, representing two presumed species, is clearly visible although it only concerns their size, and this is also reflected by their distinctness in PCA, mostly along the PC1 axis (TN versus N). Indeed, the shells from locality N03 do not differ from the ones from the two Tinos localities, representing the same presumed species. The shells of P. chia in PCA show distinctness from clade A1, but are more or less mixed with the shells of the other Pseudamnicola from the studied area.

In addition, there were no apparent differences distinguishing the shells of P. brachia (clade C) and P. pieperi (clade KAR), although PCA grouped them separately. On Rhodes Island, shells from localities R02 and R04 were characterised by their expanded apertures and differed in size and proportion from the shells in locality R06, representing another presumed species. PCA confirmed the distinctness of R01 and R02.

In the literature, the Greek Pseudamnicola species are distinguished through the application of only two criteria: shell morphology and geographic distribution (Schütt 1980). Our results certainly do not confirm those criteria. Morphometry shows some differences in the shell morphology of the particular taxa, but those differences concern no qualitative characters routinely used in species descriptions, like shape, general habitus, etc.

A similar lack of differences between the species in the characters of the female reproductive organs in Pseudamnicola was found by Szarowska and Falniowski (2011). Furthermore, the penes of the four presumed species from the islands of Andros, Tinos and Naxos are indistinguishable, as are those from the Peloponnese and Kythira Island (Szarowska and Falniowski 2011). In contrast, the penes of P. chia and P. brachia, although characteristic, cannot be used to distinguish these two species. Similar penes could also be found in the Pseudamnicola inhabiting Rhodes Island. Thus, in general, it is difficult to use penis characters to distinguish species within the Greek Pseudamnicola.

Female reproductive organ characteristics, such as receptacula, bursae copulatrix, etc., as well as penis morphology, have been found to be useful characteristics to infer phylogeny at higher levels, at least at the genus level, but certainly not at the species level (Szarowska 2006; Szarowska and Falniowski 2008). Pseudamnicola seems to be a further example of morphostatic radiation, as defined by Davis (1992), which is so common in truncatelloid snails (Wilke and Davis 2000; Wilke and Falniowski 2001; Falniowski and Szarowska 2011; Falniowski et al. 2012).

History of Pseudamnicola at the Aegean Islands

In fact, high genotypic diversity was noted on the islands of Kythira and Rhodes—two islands close to the mainland. Similarly high or even higher differences were noted between the Peloponnese populations. This may suggest that the continent offers conditions conducive to the longevity of metapopulations and may serve as a source of migrants, transported passively, most probably by birds. Two presumed species on Rhodes Island, which that are situated a few kilometres from each other, differ more from one another than each of them does from P. pieperi from the distant Karpathos Island. The relatively low genetic distances between the Cretan P. brachia and the other species despite the long distance and time of isolation of Crete are also notable. In contrast, relatively high diversity in the Andros/Tinos/Naxos complex is similar to that noted for plants by Kougioumoutzis et al. (2014). Another striking pattern is shown by the relatively close relationships between this group of islands and Khios, which could be explained by a nearly complete land bridge in the Late Pleistocene. The estimated times of divergence of Bythinella in continental Greece (Falniowski and Szarowska 2011) unexpectedly suggest that this genus has a rather short history in the studied area. We have found the same to be true in our study of Pseudamnicola.

According to geologic and climatic events, some key dates can be highlighted: the initiation of the mid-Aegean Trench split (around 12 Mya), its ending (around 9 Mya) and the Messinian crisis (around 5 Mya). The modern post-Alpine European topography in the Mediterranean emerged in the Late Tortonian (8 Mya) (Kostopoulos 2009). In the Upper Miocene (around 6 Mya), the Peloponnese and Evvoia were part of a continuous land mass with the continent (Popov et al. 2004). In the Middle-Late Pliocene (3.5–1.8 Mya), Evvoia was still a part of the continent, but the Peloponnese became separated from the continent by a broad strait (Creutzburg 1963; Dermitzakis and Papanikolaou 1981; Dermitzakis 1990; Popov et al. 2004). During the glacial maxima, the sea level was as much as 200 m lower than today (Beerli et al. 1996). During the glacial maxima in the Pleistocene, on the Greek island Andikythira (16 kya, and thus during the last glacial maximum: LGM) temperatures were some 5–8 °C below present levels, and climatic conditions were slightly drier (Digerfeldt et al. 2000; Gittenberger and Goodfriend 2006). For Bythinella from continental Greece, the estimated time of divergence did not exceed 4 Mya (Falniowski and Szarowska 2011). Schilthuizen et al. (2004) estimated a similarly short time for the Cretan Albinaria, at 3.5 Mya. In the Pliocene, some land connections disappeared due to a rise in sea level (Dermitzakis and Papanikolaou 1981; Dermitzakis 1990), but the present Peloponnese was isolated from the mainland, which coincides, in our study, with the dating of the divergence time between two of the three Peloponnese Pseudamnicola populations and the third one.

The Aegean Islands are divided into the following five groups: those associated with (i) Asia Minor (Khios and Rhodes in our study); (ii) the eastern shelf of Greece (Evvoia in our study); (iii) the Crete/Karpathos complex; (iv) the Cyclades that form a central, independent shelf (Andros, Tinos and Naxos in our study); and (v) Kythira. The central group (Cyclades) has been separated from Europe since the Middle Pleistocene (Foufopoulos and Ives 1999). Since the peak of the Wisconsin-Würm glaciation (24–10 kya), global sea level has risen 120–130 m and, in the present Aegean Islands, replaced dry land. Between 350 and 250 kya, the sea area was restricted again, and almost 50–60 % of the present Aegean Sea was land with extensive drainage systems and large lakes (Lykousis 2009). In the Late Pleistocene, the eastern Aegean Islands were connected with Anatolia, and the strait between Europe and the Cyclade-mega-island, connecting current-day Naxos, Paros, Tinos and Andros, was rather narrow (Kougioumoutzis et al. 2014). The present geography of the Aegean is no more than 8–6 kya old (Kougioumoutzis et al. 2014).

Levels of gene flow, as well as metapopulation structure in general, depend on several species-specific traits (Bohonak 1999; Bilton et al. 2001; Myers et al. 2001). The model of allopatric fragmentation and/or vicariant speciation in the Aegean Pseudamnicola does not agree with the mtCOI sequence divergence data, suggesting the relatively recent origin of the observed differentiation, which is attributable to geologic events only in some cases (see above). In the same manner, vicariance cannot explain the whole pattern in the land snail Albinaria (Douris et al. 1998, 2007) and other invertebrates and vertebrates in the Aegean (Dennis et al. 2000; Kasapidis et al. 2005). Just as in the central European Bythinella (Falniowski 1987; Falniowski et al. 1998, 1999; Szarowska 2000) and the Greek mainland Bythinella (Falniowski and Szarowska 2011), the present data did not confirm either the complete isolation of the Aegean Pseudamnicola island populations or their stability and longevity, thus confirming the second model in which a relatively young fauna is composed of more or less widely distributed taxa, with relatively high levels of gene flow among the springs they inhabit. For inhabitants of such miniature and changeable habitats, survival must depend on dynamic processes of colonisation and recolonisation events coupled with short- or (in some cases) long-distance dispersal. Pseudamnicola is not a typical oligostenothermic inhabitant of springs as it can also be found in streams, and it is somewhat amphibiotic as it can be found crawling and feeding above the water (personal observations), thus seeming capable of passive transportation by birds (for review of the literature on bird transportation, see Falniowski and Szarowska 2011).