Hydrobioid gastropods include the family Hydrobiidae Troschel, 1857 and several other families of Rissooidea s.l. that resemble Hydrobiidae in general features (Hershler and Ponder 1998). Rissooidea s.l. is one of the largest and most taxonomically challenging gastropod superfamilies (Bouchet and Rocroi 2005). Recently, Criscione and Ponder (2013) using molecular data showed that that there are two major clades encompassing taxa previously included in Rissooidea. These clades are the Rissooidea s.s. and the Truncatelloidea, the latter containing the hydrobioid families (Wilke et al. 2013).

Greek freshwater ecosystems are widely recognized as hotspots of European freshwater biodiversity (e.g. Glöer and Maassen 2009, Glöer et al. 2010, Oikonomou et al. 2012). Hydrobioids are in accordance with this observation since they are highly diverse; 77 species and subspecies belonging to 29 genera have already been recorded in Greek freshwater systems, most of them (i.e. 68.4% of the species and subspecies and 34.5% of the genera) being endemic for Greece (Bank 2006, Falniowski and Szarowska 2011a, Radea 2011, Szarowska and Falniowski 2011a, Glöer and Georgiev 2012, Georgiev 2013a, b, Radea et al. 2013). Nevertheless, the freshwater gastropod fauna of Greece remains understudied compared to other taxa (i.e. freshwater fishes) due to the complex hydrographic network of its drainages.

Almost all described Greek hydrobioid species are included in the IUCN Red List of Threatened Species (2012) and 55% of them have been classified as threatened. Graecoanatolica macedonica Radoman & Stankovic, 1978 is characterized as Extinct, and is followed by twenty four species that have been classified as Critically Endangered, five as Endangered, nine as Vulnerable, three as Near Threatened, five as Least Concern and twenty two as Data Deficient.

During 2012, several localities across Greece were sampled by the authors for hydrobioid freshwater gastropods. The sampling took place following the goals of the research project “Species on the brink of extinction” that was funded by the public benefit foundation “John S. Latsis”. The goals of this project was a) to assess and evaluate the population status of 10 freshwater snails species of Greece (9 endemics) which, according to the recent report from the International Union for Conservation of Nature (IUCN), are classified as either Extinct or Critically Endangered, b) to evaluate the status of the wetlands these species are present in and to assess the main anthropogenic regime of threats. In the network of localities sampled for the purposes of the project, we added several more localities hosting water bodies that are frequently reported in the freshwater literature of Greece or are in the vicinity of the primarily targeted localities. Therefore, we do not consider our fieldwork to be exhaustive; rather it is focused on freshwater localities that have been searched before, are frequently reported in the literature, as hosting (or having hosted) threatened species, and we complemented these localities with surrounding ones that could have served as the refuges of the threatened species.

Some of the findings of this survey as well as suggestions for the IUCN status update of some hydrobioids collected, are presented and discussed herein. Additionally, using COI sequence data generated from some specimens, we aimed to elucidate the taxonomic status of:

(i) The Greek endemic taxon of the genus Pseudamnicola Paulucci, 1878, namely Pseudamnicola (Pseudamnicola) macrostoma negropontina (Clessin, 1878). Based on the slight morphological and anatomical differences between Pseudamnicola (Pseudamnicola) macrostomanegropontina and Pseudamnicola (Pseudamnicola) macrostoma macrostoma (Küster, 1853), Szarowska et al. (2006) claimed that these taxa should be considered as distinct species. This claim was evaluated in the light of the generated sequence data.

(ii) Two new populations of the genus Graecoanatolica Radoman, 1973, which were recorded in Sterea Ellada (Voiotia). The morphological and anatomical studies were not conclusive in assigning these populations to the known Greek Graeconatolica species, Graeconatolicavegorriticola (Schütt, 1962) and Graecoanatolica macedonica, or even to a new species of Graecoanatolica. Therefore, COI sequence data were used to compare the Graecoanatolica vegorriticola specimens collected from the type locality of the species, with those of the newly located southern populations.

Overall, the current study offers new distributional data and, in the light of these, is evaluating the conservation status of some hydrobioid species of Greece. Furthermore, using newly generated COI sequence data, we are resolving taxonomic uncertainties that could not be elucidated based on morphology alone.

The freshwater localities of almost all the threatened hydrobioids of Greece were carefully sampled (Fig. 1). The snails were hand collected from stones, gravel and dead leaves (Fig. 2). Specimens were placed into vials filled with water and were transported alive to the laboratory. A portion of the specimens collected from each locality was stored in -20°C for molecular analysis, whereas others were preserved un-relaxed in 70% ethanol for morphological and anatomical studies.

Shell characters (shell height and width, apertural height and width) were measured using the micrometer of a stereomicroscope (Stemi 2000-C, Zeiss). Digital pictures were taken using a Canon EOS 1000D camera that was attached to the stereomicroscope. During this procedure, the specimens were submerged into water in order to avoid the malformation of important taxonomic features that can be caused by the long-term tissue preservation buffers.

Details of the specimens used in the molecular analyses are provided in Table 1. The entire individuals were used for total genomic DNA isolation. In total, we extracted DNA from one specimen of Pseudamnicola macrostoma macrostoma and sixteen specimens of Graecoanatolica from Pella and Voiotia.

DNA was extracted using the Purelink Genomic DNA mini kit (Invitrogen) following the manufacturers’ protocol. A fragment of the mitochondrial gene cytochrome oxidase subunit I (COI) was amplified from each specimen using the universal primers LCO1490 and HCO2198 (Folmer et al. 1994). PCR was performed in a 25 μl volume, in which 1–2 μl of template DNA was mixed with 0.2 mM dNTPs, 0.4 mM of each primer and 0.5 units of Taq polymerase. The concentration of the MgCl₂ was 3.5 mM. Thermocycling was performed in a MyCycler (Biorad) thermocycler. The cycle program comprised an initial denaturation step at 95 °C for 3 min, followed by 40 cycles of 15 s at 95 °C, 1 min at 42 °C, and 1.5 min at 72°C. The cycling was ended with 10 min sequence extension at 72 °C. Automated sequencing of both strands of the PCR amplicons was performed in a PE-ABI3740 automated sequencer (using Big-Dye terminator chemistry).

The primers in the sequencing reactions were the same as in the PCR amplifications. Sequences generated for this study were deposited in GenBank under the accession numbers provided in Table 1.

Among other findings of this study, we found Graecoanatolica vegorriticola in two new localities quite distant from all the known localities of this taxon. In our effort to properly assign these populations to the species they belong to, we realized that the morphological and anatomical studies were not conclusive. To overcome this issue, COI sequence data were used to compare the Graecoanatolica vegorriticola specimens collected from the type locality of the species, with those of the newly located southern populations. The level of COI sequence divergence (1.7%) between the population from the type locality and those from the new localities is well within the range separating conspecific populations of Hydrobiidae genera, e.g. Pyrgulopsis 0–3.44% (Hurt 2004), Grossuana 3.4% (Szarowska et al. 2007), Daphniola 1.3–2.7% (Falniowski et al. 2007), Isimerope 3% (Radea et al. 2013) and other truncatelloidean genera e.g. Austropyrgus 3–5% (Perez et al. 2005). Consequently, the above populations are conspecific and belong to Graecoanatolica vegorriticola. The genus Graecoanatolica is distributed in the Balkans (two species) and in Turkey [eight species (Radoman 1983, 1985, Kebapçi et al. 2012)]. Radoman (1985) refers that the disjunct distribution of the genus could be an evidence of a paleohydrogeographical link between Anatolia and the Balkans. However, this connection could not be verified by molecular and anatomical data as the Balkan species were reported to have gone extinct (Kebapçi et al. 2012). The discovery of fresh specimens of Graecoanatolica vegorriticola in three localities of Greece eliminates this obstacle, and the alledged paleohydrogeographical link hypothesis can now be evaluated, provided that sequence data from the Turkish species become available.

Due to insufficient morphological and anatomical differentiation, the nominal subspecies Pseudamnicola macrostoma macrostoma (Küster, 1853) and Pseudamnicola macrostoma negropontina cannot be discriminated. However, Szarowska et al. (2006) supported that these taxa should be considered as distinct species. This claim was evaluated in the light of the generated sequence data from the newly collected specimens. The sequence divergence separating Pseudamnicola macrostoma macrostoma and Pseudamnicola macrostoma negropontina is 5.8% and this level of sequence divergence falls within the range 3.7–7.0% [Szarowska and Falniowski (2011), table 5 and p. 126] that separates Greek Pseudamnicola species. Therefore, it can be claimed that based on the COI sequence divergence levels, additional data exist to support the view that, despite the morphological and anatomical similarity between the two taxa, a species statusshould be assignedto Pseudamnicola macrostoma negropontina, as Szarowska et al. (2006) suggested.

During the field survey undertaken for this study, several threatened taxa, such as Graecoanatolica vegorriticola, Pseudamnicola negropontina, Pseudamnicola pieperi, Pseudobithynia eubooensis and Pseudoislamia balcanica, were recorded from new localities. Trichonia trichonica, which has been considered extinct from its type locality for the last twenty eight years (Albrecht et al. 2006), was re-discovered, whereas the presence of Daphniola exigua, Graecoanatolica vegorriticola, Marstoniopsis graeca, Pseudamnicola pieperi and Pseudobithynia trichonis in their type localities was verified. These findings combined with the recent discovery of the new endemic genus and species, Isimeropesemele (Radea et al. 2013), besides confirming the crucial role of the Greek freshwater systems in shaping Europe’s freshwater biodiversity (Glöer and Maassen 2009, Glöer et al. 2010), they also highlight two major issues, a) the Greek freshwater systems have yet a lot to offer to this diversity if comprehensively studied, and b) the IUCN status of some Greek endemic hydrobioids needs to be updated. Towards the latter issue we support that the new data derived from this survey allow us to propose transfers between categories for some species included in the IUCN Red List Threatened Species (2012) ver.3.1 (Table 2). These transfer proposals are based on the newly acquired knowledge regarding the distributional ranges of the species (Graecoanatolica vegorriticola, Pseudoislamia balcanica and Trichonia trichonica) as well as to the elucidation of the standing taxonomic confusion of certain species i.e. newly split (Pseudamnicola negropontina) and newly described (Isimerope semele). We followed the IUCN guidelines regarding the definition of the term “location”: “a geographically or ecologically distinct area in which a single event (e.g. pollution) will soon affect all individuals of the taxon present” (IUCN 2011).

Graecoanatolica vegorriticola. Our findings indicate that none of the criteria of the category Critically Endangered are met since the extent of occurrence (EOO) and the area of occupancy (AOO) become >100 km² and >10 km², respectively. Therefore, this species may be down-listed to the category Endangered if the criteria of the category Critically Endangered continue to not be met for the next five years. Additionally, (a) and b(iii) are met because the number of locations is ≤ 5 and a continuing decline is observed in the quality of the habitat, respectively; c(iv) is also met because extreme fluctuation in the number of mature individuals has been recorded (Hauffe et al. 2011).

Pseudoislamia balcanica. The discovery of this species in a new locality increases the extent of its occurrence (EOO), which becomes >100 km². Therefore, Pseudoislamia balcanica may be down- listed to the category Endangered if it continues to thrive in other locality(ies), apart from Lake Trichonis, at least for the next five years. Additionally, (a) and b(iii) are met because the number of locations is ≤ 5 and a continuing decline is observed in the quality of the habitat, respectively.

Trichonia trichonica. We re-discovered this species in Lake Trichonis. On the contrary, no single specimen or even empty shells were recorded in Krya’s spring (Lake Pamvotis) due to the water intake built on it. Therefore, the criterion B1, the extent of occurrence (EOO) <100 km², is met for Trichonia trichonica. Additionally, (a) and b(iii) are met because the number of locations is 1 and a continuing decline is observed in the quality of the habitat, respectively.

Pseudamnicola negropontina has not yet been evaluated because it is now elevated to species level (newly split). The area of occupancy of this species is < 10 km² and, consequently, the criterion B2 is met. Additionally, (a) and b(ii, iii) are met because the number of locations is 1 and a continuing decline is observed in the area of occupancy and the quality of the habitat, respectively.

Isimerope semele has not yet been evaluated because it is a newly described species (newly described). The extent of occurrence of this species is 100 km²<EOO<5000 km² and therefore the criterion B1 for the category Endangered is met. Additionally, (a) and b(iii) are met because the current distribution of the species is severely fragmented and a continuing decline is observed in the quality of the habitat, respectively.

During the field survey we ascertained that many of the “hydrobioid” localities in Greece are heavily influenced by various human activities such as tourism, agriculture, livestock, industry, housing development and forestry. Thus, a decline or even loss of local freshwater gastropods is expected and, in some cases, it has already been reported (Ryan and Griffiths 2001, Szarowska and Falniowski 2004, Albrecht et al. 2006, Regnier et al. 2009).

Against the loss of hydrobioids due to the declining number of suitable habitats, a taxonomically accurate record of taxa, especially for those thriving in springs and spring brooks (crenobionts), will contribute significantly in assigning high conservation priorities (Arconada and Ramos 2001, Haase 2003). Without serious and effective intervention it is only a matter of time before the vast majority of the unprotected hydrobioids we recorded in this study become extinct.