The dwarf pond snail Galba truncatula (O.F. Müller, 1774) is a widespread freshwater species and a key intermediate host of Fasciola spp. Despite its ecological and medical significance, the evolutionary structure of its populations remains incompletely resolved. Using 78 mitochondrial COI sequences, including newly obtained material from Eastern Europe, Central Asia, and Caucasus, we reconstructed the phylogeographic pattern of Galba truncatula across Western Palearctic. Our analyses reveal the presence of three distinct phylogenetic lineages. A deeply divergent group, represented by a small number of sequences, occurs sympatrically with the main clade in Western Europe. The main clade, comprising the majority of sequences, is split into two geographically structured subclades: a western lineage (Western Europe and North Africa) and an eastern lineage (Eastern Europe, Caucasus, and Central Asia). Both major lineages are represented in South America, suggesting at least two independent colonization events. We propose that the initial divergence between the eastern and western lineages likely began in the early Pleistocene, whereas their present-day distribution may have been shaped by isolation during the Last Glacial Maximum.

COI gene, cryptic diversity, fascioliasis vector, freshwater molluscs, invasive species, Palearctic, phylogeography, South America

The dwarf pond snail, Galba truncatula (O.F. Müller, 1774), is a widely distributed freshwater and amphibious gastropod across the Palaearctic and adjacent regions and is recognized as a key intermediate host for a variety of some infections caused by parasites (Kruglov 2005). Notably, it serves as the primary vector for liver flukes of the genus Fasciola Lamarck, 1799, which are responsible for fascioliasis in both livestock and humans (Kruglov 2005; Artigas et al. 2011; Králová-Hromadová et al. 2011; Mahulu et al. 2019). Galba truncatula is also known from South America, where it was most likely introduced (Standley et al. 2013; Vázquez et al. 2022). A less widely accepted hypothesis, however, suggests the opposite scenario that the species was introduced from the New World to the Old World, followed by its rapid spread there (Correa et al. 2010).

Galba truncatula is one of the few freshwater molluscs that not only inhabits continental aquatic habitats from tundra to steppe, but has also successfully colonized Atlantic islands and archipelagos, including Iceland and the Faroe Islands (Armitage and McMillan 1963; Carlsson 2001; Kruglov 2005; Vinarski et al. 2021). It has further been recorded from islands in the Barents Sea, demonstrating its capacity as a pioneering species capable of colonizing newly available habitats (Nekhaev 2021; Vinarski et al. 2021b; Bespalaya et al. 2022).

Widespread species often exhibit complex patterns of phylogenetic structure and may, in fact, represent species complexes composed of cryptic or pseudocryptic taxa whose distribution can reflect past geological events (Vinarski et al. 2017; Vinarski et al. 2022; Aksenova et al. 2024; Novikov et al. 2025). Conversely, the remarkable dispersal ability of Galba truncatula, coupled with its well-documented morphological plasticity (Chapuis et al. 2007), may contribute to a comparatively homogenized genetic structure across its extensive range.

In recent decades, several species conchologically similar to Galba truncatula have been described or re-described from various parts of Eurasia, many of which exhibit narrow and often localized distributions (Izzatullaev et al. 1983; Kruglov 2005; Vinarski and Kantor 2016). This pattern could suggest that the broadly defined dwarf pond snail may comprise multiple evolutionary lineages across its range. However, such taxonomic distinctions are primarily based on variations in shell morphology and reproductive anatomy, which may themselves reflect ecophenotypic plasticity rather than true genetic divergence, as demonstrated in other widespread freshwater molluscs (Bolotov et al. 2013; Aksenova et al. 2018; Vinarski et al. 2021a).

Although the dwarf pond snail is frequently targeted in phylogenetic and DNA barcoding studies (Hurtrez-Boussès et al. 2010; Standley et al. 2013; Alda et al. 2018; Schniebs et al. 2018), most of this research has focused on populations from Western Europe or the species’ invasive range, while vast regions of Eastern Europe, Asia and the Caucasus remain largely understudied. To date, no comprehensive attempt has been made to assess the population structure of Galba truncatula across its full distribution.

In this study, we aim to evaluate intraspecific genetic diversity in Galba truncatula sensu lato by integrating newly generated COI gene sequences with publicly available data, providing the first broad-scale assessment of phylogeographic patterns in this species.

Phylogenetic trees reconstructed using both maximum likelihood and Bayesian inference methods revealed largely congruent topologies for sequences identified as Galba truncatula (Fig. 2) (Suppl. material 2). In both trees, three sequences from the northern part of Western Europe formed a distinct clade separate from the remainder of the dataset. All other sequences clustered into a single major clade, which further subdivided into two well-supported subclades.

The first subclade (hereafter referred to as the “eastern clade”) comprises sequences from Asia, the Caucasus, and Eastern Europe. The second (“western clade”) includes the remaining sequences from Western Europe and, in part, from North Africa. Further internal subdivisions were observed but corresponded to comparatively shorter genetic distances.

The TCS haplotype network revealed a similar pattern of phylogeographic structuring (Fig. 3). A total of 36 haplotypes were identified, forming a well-structured network. A distinct group consisting of three sequences from Northern Europe (representing two haplotypes) was separated from the rest of the network by 36 mutational steps.

Figure 2. 

Bayesian Inference Tree and map of georeferenced records of Galba truncatula from Palearctic region. Values of posterior probability less than 0.7 are omitted. The extent of the maximum ice sheet and Alpine glaciation during the Last Glacial Maximum is indicated in blue based on published sources (Svendsen et al. 2004; Hughes and Woodward 2017).

Figure 3. 

TCS haplotype network of Galba truncatula.

Within the main haplogroup, two subgroups were apparent: a western and an eastern subgroup, separated by 18 mutational steps. Notably, both subgroups were represented in the invasive range of the species; however, invasive populations did not share any haplotypes with those from the native range.

The eastern clade comprised 18 haplotypes. Haplotypes from the Caucasus and the Himalayas were unique to these respective regions, although they differed from each other by only a few mutational steps. Galba truncatula populations from the Tien Shan shared haplotypes with Eastern Europe, which exhibited the highest haplotype diversity within the eastern group. One haplotype from South America was also nested within the eastern clade.

The western clade exhibited a more limited geographic distribution and included 12 haplotypes. Three haplotypes were found in North Africa and three in South America, while the remainder were restricted to Western Europe. Notably, North African populations shared no haplotypes with European populations.

AMOVA revealed a pronounced genetic structuring differentiating the eastern and western clades. A substantial proportion of total genetic variance (70.6%) was attributable to differences between clades (Phi statistic = 0.707, p < 0.001), while the remaining 29.4% reflected variation within clades.

Nucleotide diversity, calculated excluding invasive populations, was higher in the western clade (π = 2.0%) than in the eastern clade (π = 0.97%). Within Western Europe alone (excluding North African populations), nucleotide diversity remained elevated (π = 1.9%).

One of the most intriguing findings of our study is the discovery of two deeply divergent phylogenetic lineages of Galba truncatula occurring in sympatry within Western Europe. These lineages are separated by a substantial number of mutational steps, suggesting long-term evolutionary separation. Unfortunately, one of the groups is represented by only three sequences retrieved from GenBank, and no morphological data are available for these specimens.

Remarkably, representatives of both divergent lineages have previously been used concurrently in broader phylogenetic studies of the Lymnaeidae, where their distinctiveness appeared to be overlooked or considered taxonomically insignificant (Schniebs et al. 2018; Mahulu et al. 2019; Saito 2022).

Although both divergent European lineages may represent distinct species, formal taxonomic conclusions are beyond the scope of this study and are hindered by the absence of corresponding morphological data.

The inclusion of topotypic material of Galba almaatina allowed us to reject the hypothesis of its species-level distinctiveness and hence consider that species to be a synonym of Galba truncatula. In addition, several other Galba species: G. goupili (Moquin-Tandon, 1855), G. subangulata (Roffiaen, 1868), G. oblonga (Puton, 1847), and G. sibirica (Westerlund, 1885) – have been previously reported from Eastern Europe and northern Asia (Kruglov 2005; Vinarski and Kantor 2016). These taxa have traditionally been considered as separate species by Soviet and Russian authors, based primarily on subtle differences in shell proportions. Our results do not support the presence of more than one Galba species in aforementioned areas. Nevertheless, a robust taxonomic revision would require the inclusion of topotypic material for these nominal species and an integrative approach combining molecular, morphological, and ecological data.

The largest clade of Galba truncatula is subdivided into two groups that are strongly supported by AMOVA. However, although the calculated p-distance indicates deep intraspecific divergence, it remains below the species-level threshold commonly accepted for the family Lymnaeidae, where a COI divergence value of 0.05 or higher is typically proposed as indicative of species-level separation (Pfenninger et al. 2006). A study of the closely related species Galba cubensis further demonstrated that even at higher levels of COI divergence (e.g., 0.059), nuclear ITS2 variability remained low (Ferreira et al. 2021). These findings suggest that the observed genetic distance does not provide sufficient evidence to justify species delimitation.

Both subclades display a clear geographic pattern in their distribution. A similar east–west phylogeographic split has been observed in several other widely distributed plant and animal species across Eastern Europe and Siberia (Taberlet et al. 1998; Hewitt 1999; Schmitt 2007). This pattern is typically attributed to population isolation during the Pleistocene, when Europe and western Siberia experienced repeated cycles of glaciation (Webb and Bartlein 1992; Svendsen et al. 2004; Vandenberghe et al. 2014; Hošek et al. 2024).

The expansion of the continental ice sheets was accompanied by the growth of alpine glaciers in European mountain systems, including extensive glaciation in the Mediterranean region. The most prominent of these was the Alpine glaciation, which extended well beyond the mountain range itself (Hughes and Woodward 2017; Hošek et al. 2024). The southward advance of the Scandinavian ice sheet, together with mountain glaciations, formed a barrier separating Western Europe (which was only partially glaciated) from the region encompassing Eastern Europe and Siberia (Svendsen et al. 2004; Fig. 2). These events were associated with a general decline in global temperatures and the development of permafrost in areas not directly covered by ice (Vandenberghe et al. 2014).

Although the current range of Galba truncatula extends into northern regions within the tundra zone (Nekhaev 2021; Vinarski et al. 2021), the species is absent from true Arctic or high-altitude periglacial landscapes (Coulson et al. 2014), which share ecological characteristics with much of glacial-period Europe. This suggests that during Pleistocene glacial cycles, the distribution of the dwarf pond snail likely contracted alongside boreal communities.

We hypothesize that the divergence between the two major linages may have begun as early as the early Pleistocene, as has been proposed for other organisms with comparable levels of genetic differentiation (Taberlet et al. 1998). The Last Glacial Maximum, which peaked between approximately 25 and 17 ka (Svendsen et al. 2004), likely reinforced this separation by creating additional barriers to gene flow and shaping the present-day distribution of these lineages (Fig. 2).

A notable feature of the current distribution is the apparent lack of admixture between the two geographic groups, despite the species’ high dispersal capacity. This suggests long-term maintenance of lineage separation, possibly through ecological or reproductive isolation mechanisms.

This pattern is also observed in several other groups of terrestrial and freshwater animals (Taberlet et al. 1998; Vainio and Väinölä 2003; Schmitt 2007). A commonly proposed explanation is ecological competition for an available niche, with the group that first establishes a stable population typically outcompeting later arrivals (Taberlet et al. 1998). However, in the case of Galba truncatula, this hypothesis is challenged by our finding of two highly divergent phylogeographic groups co-occurring in sympatry in Western Europe.

Furthermore, both phylogeographic lineages of Galba truncatula appear to share parts of their ranges with morphologically similar, ecologically analogous species such as Galba cubensis (Pfeiffer, 1839) and Galba schirazensis (Küster, 1833), both of which have recently expanded their distributions into Eurasia (Schniebs et al. 2018; Tahirova et al. 2025). This highlights the potential complexity of ecological interactions and underscores the need for integrative approaches in understanding the mechanisms maintaining species and lineage boundaries in this group.

We cannot rule out the possibility that the apparent lack of admixture between the two geographic linages may be an artifact of limited sampling. However, if gene flow does occur, its extent appears to be minimal.

Our data do not support the existence of ancient refugia for Galba truncatula in the Caucasus or Eastern Europe, despite the fact that these regions provided suitable conditions for the persistence of boreal organisms, and even entire boreal communities, during Pleistocene glaciations (Copilaş-Ciocianu and Petrusek 2017; Richter et al. 2020; Hošek et al. 2024; Bikashvili et al. 2025).

We found substantially higher haplotype diversity in Eurasian populations compared to those from South America. The low genetic diversity observed in the New World is consistent with a founder effect and likely reflects a population bottleneck during colonization, possibly due to a limited number of introduction events or dispersal vectors.

This pattern does not support the hypothesis of a South American origin for Galba truncatula, previously proposed by Correa et al. (2010). Instead, our data suggest that dwarf pond snail colonized South America independently on at least two occasions, involving representatives of both the eastern and western clades. This scenario is supported by the coexistence of both lineages in South America, despite their geographic segregation in Eurasia.

Multiple introductions of genetically divergent lineages have been previously documented in other pulmonate snails, such as the New World species Physella acuta and Planorbella duryi (Albrechtet al. 2025; Nekhaev et al. 2024). Our results suggest that similar processes can occur in the opposite direction – from Eurasia into the Americas.

Our study reveals a previously unrecognized phylogeographic complexity within Galba truncatula. We identified three divergent mitochondrial lineages, including a deeply separated group co-occurring with the main clade in Western Europe. The primary division between eastern and western clades likely originated in the early Pleistocene and was reinforced by glacial isolation during the Last Glacial Maximum.

The absence of haplotype sharing between lineages, despite their overlapping ranges and high dispersal potential, points to long-term evolutionary separation. The presence of both clades in South America suggests multiple independent introduction events. These findings have implications for taxonomy, epidemiology, and the reconstruction of postglacial biogeographic histories in freshwater taxa.

We are thankful to Pavel Kijashko for the possibility to access ZIN collections. The photographs of holotype of Lymnaea almaatina were taken using facilities of Centre “Taxon” of ZIN.

The study was supported by Science Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan (Grant No. AP22783298). Molecular analysis of snails (DNA extraction, PCR, and sequencing) was supported by the Russian Science Foundation (project No. 21-74-10155).

Ivan Nekhaev – Conceptualization, Visualization, Funding acquisition, Investigation, Methodology, Software; Writing – original draft; Anel Ishayeva – Data curation, Formal analysis; Amina Omarova ­– Data curation, Formal analysis, Software; Irina Khrebtova – Investigation, Resources; Alexander Kondakov ­– Methodology, Investigation, Resources; Leonid Kim – Investigation; Olga Aksenova – Investigation, Resources, Writing – review & editing.