The fresh- and brackish water gastropods of Iran have been studied for a long time (Issel 1865; Forcart 1935; Biggs 1936, 1937, 1971; Starmühlner and Edlauer 1957; Starmühlner 1965; Schütt 1973; Ghadirian and Hoghooghi 1973; Eliazian et al. 1979; Massoud and Hedayeti-Far 1979; Mansoorian 1986, 1992, 1994, 2001; Glöer and Pešić 2006, 2009, 2012; Ghasemi et al. 2011; Ektefa et al. 2013; Imani-Baran 2016). However, these studies are not extensive, and most have focused on the medical and parasitological aspects of snails as potential hosts of parasitic trematodes. Scarce studies have reported the few representatives of Truncatelloidea recorded from Iran so far. Among them, seven species represent the family Bithyniidae E. J. Gray, 1857 [in Turton 1857] (Glöer and Pešić 2012), three the family Stenothyridae Tryon, 1866 (Glöer and Pešić 2012), and one the family CochliopidaeTryon 1866 (Glöer and Pešić 2012). Twelve species belong to the family Hydrobiidae W. Stimpson, 1865 (Glöer and Pešić 2012): two of them are from the brackishwater subfamily Hydrobiinae W. Stimpson, 1865, and eight are from the subfamily Pseudamnicolinae Radoman, 1977. In addition to an enigmatic valvatiform minute shell presented by Starmühlner and Edlauer (1957) and identified by them as Horatia (Hauffenia) erythropomatia Hauffen, 1856 (neither the shell morphology nor the geographic distribution of this Slovenian taxon support this identification), Starmühlner and Edlauer (1957) described another minute but ovate-conical gastropod Frauenfeldia elbursensis Starmühlner & Edlauer, 1957. However, Frauenfeldia Clessin, 1878 is a junior homonym, thus its replacement name is Graziana Radoman, 1975. Glöer and Pešić (2012) listed it as Belgrandiella elbursensis (Starmühlner & Edlauer, 1957), then later Glöer (2022) assigned it to the genus Pseudamnicola Paulucci, 1878. This minute (about 2 mm high) inhabitant of a small mountainous stream in Iran (Starmühlner and Edlauer 1957), whose shell resembles Graziana, cannot be assigned confidently to any truncatelloid genus due to the lack of anatomical or molecular data. Recently, Fatemi et al. (2019) recorded the first representative of the stygobiont family Moitessieriidae Bourguignat, 1864 – Trogloiranica tashanica Fatemi, Malek-Hosseini, Falniowski, Hofman, Kuntner & Grego, 2019, type species of the genus Trogloiranica Fatemi, Malek-Hosseini, Falniowski, Hofman, Kuntner & Grego, 2019.

Apart from Trogloiranica, there is no information available on stygobiont or other subterranean snails in Iran. Our literature survey revealed no records of Iranian truncatelloid snails inhabiting springs. Therefore, the few specimens of minute truncatelloid gastropods collected in Tuveh Spring, Iran, are noteworthy. The snails lacked both eyes and pigmentation in their soft parts, clearly indicating troglomorphic adaptations (Culver 2012; Culver and Pipan 2009, 2014). These specimens were most likely flushed out of the subterranean aquifer by ephemeral waters. In this paper, we describe these snails.

Herein, we describe a new stygobiotic genus and two new species of the gastropod family Hydrobiidae from groundwaters of Iran using morphology as well as (for one of the two species) COI, H3 and 28S sequence data. We conclude that this aquifer in Zagros mountains harbours more stygobiotic species than the three Iranian cave fishes previously reported from this spring (and from some other localities connected to this large aquifer).

Specimens were collected by hand (30 May 2019) at Tuveh spring, close to Tuveh village, Andimeshk, Khuzestan Province, 32°48'47"N, 48°43'6"E, 495 m a.s.l. (Fig. 1).

Shells were photographed with a Canon EOS 50D digital camera, under a Nikon SMZ18 microscope. Dissections were done under a Nikon SMZ18 microscope with dark field. Morphometric parameters of the shell were measured by one person using ImageJ image analysis software (Rueden et al. 2017). Penises were photographed under a Motic B3 Professional microscope with dark field. Morphological terminology followed Hershler and Ponder (1998).

Scanning Electron Microscopy (SEM) was used to image the surface of the shell. The specimens were cleaned in distilled water to remove any remaining mud and particles using a sonicator. The shells were then washed with distilled water, air-dried, and mounted on an aluminum stub using double-sided carbon tape. In the next step, shells were fixed on a holder and coated with a 30-nm layer of gold. Electron micrographs were generated utilizing advanced imaging technology through the operation of a GAOL, GSMS 400 LV scanning electron microscope specifically configured to operate in back-scattering mode. Additionally, electron micrographs were also obtained using a Cambridge Stereo Scan Mark 2A (15 kV) machine located at Shiraz University Electron Microscope Center, Iran (Echreshavi et al. 2021). The radulae were extracted with Clorox, applying the techniques described by Falniowski (1990) and photographed using a HITACHI S-4700 scanning electron microscope.

For molecular analysis, snails were put in fresh 80% analytically pure ethanol and kept at -20 °C temperature in a freezer. DNA was extracted from whole specimens. Tissues were hydrated in tris‑EDTA (TE) buffer (3 × 10 min); total genomic DNA was extracted with the Sherlock extraction kit (A&A Biotechnology), and the final product was dissolved in 20 μL of TE buffer. The extracted DNA was stored at -80 °C at the Department of Malacology, Institute of Zoology and Biomedical Research, Jagiellonian University in Kraków (Poland). A total of three markers were analyzed: Folmer’s fragment of the mitochondrial cytochrome c oxidase subunit I (COI) (Folmer et al. 1994) and the nuclear histone 3 (H3) (Colgan et al. 2000) were sequenced using the same PCR conditions, primers and Sanger sequencing method as in Szarowska et al. (2016). For improved accuracy, the same two markers as well as Veronik’s fragment of the 28S ribosomal RNA gene (Verovnik et al. 2005) were subsequently amplified and sequenced using Nanopore as in Navarro-Mayoral et al. (2024).

Phylogenetic analyses of the COI and H3 markers were executed by addition of sequences available from the GenBank repository (Table 1), selected basing on comparisons using the BOLD Systems. Sequences were initially aligned using MUSCLE (Edgar 2004) as implemented within MEGA 7 (Kumar et al. 2016), and then visually checked in Bioedit 7.1.3.0 (Hall 1999). Uncorrected p‑distances were calculated in MEGA 7. The estimation of the proportion of invariant sites and the saturation tests for entire data sets (Xia 2000; Xia et al. 2003) were performed using DAMBE (Xia 2018). The data were analysed using approaches based on Bayesian inference (BI) and maximum likelihood (ML). The appropriate substitution models (HKY+I+G for COI and HKY+I+G for H3) for ML analysis were selected using the jModelTest2 via the CIPRES Science Gateway (Miller et al. 2010). The ML analysis was conducted in RAxML‑NG v. 0.8.0 (Kozlov et al. 2019) using the web service available at https://raxml‑ng.vital‑it.ch/, with 10 random and 10 parsimony-starting trees. For the BI analysis MrBayes v. 3.2.7a (Ronquist et al. 2012) was used with defaults priors. Two simultaneous analyses were performed, each with 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 the trees were discarded as a burn‑in. The analyses were summarized as a 50% majority‑rule consensus tree. Convergence of the runs was checked in TRACER v.1.7.1 (Rambaut et al. 2018), and all effective sample size exceeded 200, the proposed measure of good sample mixing. FigTree v. 1.4.4 (Rambaut 2010) was used to visualize the trees. Three species delimitation methods were performed: Poisson Tree Processes (PTP) (Zhang et al. 2013), Automatic Barcode Gap Discovery (ABGD) (Puillandre et al. 2011) and Assemble Species by Automatic Partitioning (ASAP) (Puillandre et al. 2021). The PTP approach was run using the web server https://species.h-its.org/ptp/, with 100,000 MCMC generations, 100 thinning and 0.1 burn-in. We used the RAXML output phylogenetic tree. The ABGD approach used the web server (https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html) with the default parameters. The ASAP approach was run using the web server (https://bioinfo.mnhn.fr/abi/public/asap/) according to simple distance (p-distances). The programmes Fastachar (Merckelbach and Borges 2020) and DeSignate (Hütter et al. 2020) were used to distinguish particular species from other species, by determining Molecular Diagnostic Characters (MDCs) for COI. Two types of characters were accepted: at binary positions (the character state in the query group is different from the uniform character state of the reference group) and at asymmetric positions (the character state in the query group is different from the non-uniform character state of the reference group).

Abbreviations:

AOO Area of occupancy

EOO Extent of occurrence

NHMW Natural History Museum Vienna, Austria

ZMUJ Department of Malacology of Jagiellonian University

JG Collection Jozef Grego, Slovakia

This study provided additional data on the subterranean fauna of Iran. Iranobelgrandia persica sp. nov. represents the second stygobiotic gastropod species from Iranian groundwater confirmed by molecular data, whereas the third species, Pseudamnicola stygopersica, is currently known only from a few empty shells. Further studies across a wider area of Iran, particularly in the karstic regions of the Zagros Mountains, are needed to improve our understanding of the country’s stygofauna. Such research will be essential for clarifying the phylogenetic relationships between Iranian subterranean fauna and other stygobiotic faunas worldwide, as well as for understanding the evolutionary history of the colonization of Iranian subterranean habitats by these organisms. Molecular trees clearly indicate close relationships between our newly described Iranobelgrandia and three European genera: Belgrandia, Dalmatinella and Montenegrospeum, as well as with Martensamnicola from Tajikistan. The type locality of Iranobelgrandia is situated far from both the type locality of Martensamnicola and the Balkans. Moreover, there is no basis for closer morphological comparison with Martensamnicola; therefore, broader taxonomic and phylogeographic conclusions would be unjustified. Nevertheless, the genetic distance and the morphological diagnoses characterising Iranobelgrandia strongly suggest that it represents a distinct genus. It is also clear that the representatives of Belgrandia and Dalmatinella belong to the same clade. However, in the GenBank there are two sequences of “Belgrandia”, phylogenetically far one from the other, and none of them representing the type species of Belgrandia. Thus, the sequence of Cyclostoma gibbum Draparnaud, 1805 is badly needed, before a decision of synonymizing Dalmatinella, or even Montenegrospeum, with Belgrandia can be made.

The female reproductive organs, as well as the penises of Belgrandia (Giusti and Pezzoli 1972, 1980; Haase 2000; Boeters and Falkner 2009), Dalmatinella (Radoman 1974, 1983; Szarowska 2006; Falniowski and Szarowska 2013; Beran et al. 2021) and Montenegrospeum (Pešić and Glöer 2013; Falniowski et al. 2013), are similar to the ones of Iranobelgrandia. Thus, morphology and molecules yield congruent pictures of phylogenetic relationships.

The finding of a snail phylogenetically closely related to the European Belgrandiinae in Western Iran was somewhat unexpected. There are no records of these snails east of the Balkans. The freshwater truncatelloid fauna of Turkey has been rather extensively studied (e.g., Radoman 1983; Yildirim et al. 2006; Çaĝlan et al. 2012; Gürlek 2017; Odabaşi et al. 2019), but no representative of the genera closely related to Iranobelgrandia has been found. However, considering the female reproductive organs and penis, although not the shell habitus (the latter hardly useful in phylogeny reconstruction in the Truncatelloidea, cf. e.g. Falniowski 2018), Turcorientalia Radoman, 1973 with its type species T. anatolica Radoman, 1973, described from the small spring Yarışlı (Yercey in Radoman 1973, 1983) near Lake Yarışlı, above the road Yesilova-Burdur in Turkey (Radoman 1973), may be closely related with Iranobelgrandia. In the current absence of molecular data on Turcorientalia, we can only speculate about this possible relationship.