Two new stygobiotic species of Horatia Bourguignat, 1887 (Hydrobiidae) from Croatia

In this paper we describe two new species of the freshwater snails of genus Horatia . A new stygobiotic species of Horatia Bourguignat, 1887 is described from Izvor Beguša in Croatia. It occurs in sympatry with the crenobiotic H. klecakiana Bourguignat, 1887, but is morphologically and molecularly distinct. It is characterized by the terminal part of the body whorl separated from the columella, and neither eyes nor any pigment on the soft parts. It is a stygobiont gastropod, known so far only from one living specimen and several empty shells, thus its soft part morphology and anatomy remain unknown. Another new species of stygobiotic Horatia was found inside the cave Mali Rumin, its description is based solely on numerous empty shells from the cave sediments.

In 2020 we collected one live specimen (Figs 3, 4A) and several empty shells of Horatia in Izvor Ruda Beguša, cave just above the spring zone and in the spring lake, sieved from sandy sediment at the spring and cave bottom, 13 km ESE of Sinj, Split District, Croatia. It occurred in sympatry with a few living specimens of H. klecakiana. The specimen was evidently a stygobiont, with neither eyes nor pigment, and with the characteristic shell, evidently different from the ones found in the other species of Horatia. Another stygobiotic species, represented by numerous empty shells and shell fragments was found inside the cave Mali Rumin, Split District, Croatia (in summer 2020), and earlier in the active spring sediments in the same locality (in spring 2017).   The snails were collected from the sediment with a 500 μm sieve and fixed in 80% analytically pure ethanol, replaced two times, and kept in -20 °C temperature in a refrigerator. The shells were photographed with a Canon EOS 50D digital camera, under a Nikon SMZ18 microscope with dark field; measurements of the shell were taken using IMAGEJ image analysis software (Rueden et al. 2017).
DNA was extracted from whole specimens; tissues were hydrated in tris-EDTA (TE) buffer (3 × 10 min); then 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).
Mitochondrial cytochrome oxidase subunit I (COI) locus was sequenced. Details of PCR conditions, primers used and sequencing were given in Szarowska et al. (2016). Sequences were initially aligned in the MUSCLE (Edgar 2004) Programme implemented in MEGA 7 (Kumar et al. 2016) and then 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 test for entire data sets (Xia 2000;Xia et al. 2003) were performed using DAMBE (Xia 2013). In the phylogenetic analysis, additional sequences from GenBank were used as reference ( Table 1). The data were analysed using approaches based on Bayesian Inference (BI) and Maximum Likelihood (ML). In the BI analysis, the GTR + I + Γ model of nucleotide substitution was applied. Model was selected using MRMODELTEST 2.3 (Nylander 2004). The analyses were run using MRBAYES v. 3.2.3 (Ronquist et al. 2012) with default of most 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 burn-in. The  (Miller et al. 2010). We applied the GTR model which is the only nucleotide substitution model implemented in RAxML, whose parameters were estimated by RAxML (Stamatakis 2014). For comparison purposes the pictures of type material of taxa described by Bourguignat, 1887, were used. The pictures were kindly provided by Emmanuel Tardy (MHNG). Diagnosis. Shell minute, valvatoid, distinguished from the other species of Horatia by its body whorl separated at its terminal sector from the penultimate one (scalarity at this part), the circular and complete peristome and extremely wide umbilicus showing earlier whorls inside.  (Fig. 4B) 2.14 mm high and 1.62 mm broad, valvatoid, whitish, translucent, rather thin-walled, consisted of about 3 ½ whorls, growing rapidly and separated by moderately deep suture, more prominent closer to the aperture: the terminal part of the body whorl completely separated from the penultimate one. Spire low and narrow, body whorl large. Aperture prosocline, circular in shape, peristome complete and separated from the columella, swollen, umbilicus extremely wide, with  Table 2. Soft parts morphology and anatomy. The body white, pigmentless, with no eyes. The anatomy unknown.

Description. Shell
Derivatio nominis. The specific epithet ozimeci refers to our friend Mr.sc. Roman Ozimec, a biospeleologist from Bast, Croatia, deeply devoted to the study and protection of the subterranean habitats in the Dinarides and Balkans.
Distribution and habitat. Known from the type locality. The type locality is a karst spring lake surrounded by three outflow caves intermittently draining the karst conduit at high water saturation. The spring draining water from sinkholes at Buško Jezero (Bosna and Hercegovina) and supports the river Ruda, a left tributary of Cetina River. The following Hydrobiidae species were detected in the habitat: Horatia klecakiana, Orientalina curta germari (Frauenfeld, 1863), Montenegrospeum sketi Grego & Glöer, 2018, Kerkia jadertina sinjana (Kuščer, 1933).
Molecular distinctness and relationships of Horatia ozimeci. We obtained eight new sequences of COI (409 bp, GenBank Accession Numbers MW448545-MW448552). The tests by Xia et al. (2003) revealed no saturation. The topologies of the resulting phylograms were identical in both the ML and BI. All the seven sequences of H. klecakiana, collected at five localities, were identical. H. ozimeci formed a sister clade with H. klecakiana (bootstrap support 100%, Bayesian probability 1.0), confirming the congenerity of the two taxa (Fig. 6). Within genus Horatia the p-distance between the taxa was 0.074. This well supported clade belongs to the Horatiinae, subfamily of Hydrobiidae. Deeper relationships within the Horatiinae remain unresolved, because of the lack of acceptable support for deeper nodes, which is typical of the phylograms based on COI. Diagnosis. Shell minute, trochiform, distinguished from the other species of Horatia by its very wide umbilicus showing earlier whorls inside. From the H. ozimeci s. nov. distinguished by higher conical spire and suture reaching the aperture, as well by more swollen protoconch and by slight sinuation at labral columellar margin. From stygobiotic cf. Horatia knorri different by much wider umbilicus and by aperture shape less declined from the columella in its labral profile.

Horatia stygorumina
Description. Shell (Fig. 4F) 2.08 mm high and 1.57 mm broad, conical, fresh shells milky white and translucent, with 3 rounded inflated whorls and deep suture,  Derivatio nominis. The specific epithet stygorumina is derived from the stygobiont habitat and from the type locality: cave Mali Rumin also referring to the name of the nearby settlement Rumin.
Distribution and habitat. Known only from the type locality, where empty shells can be found in the cave sediments, as well as in the sandy sediments of the intermittent spring. The shells are washed out from their stygobiont habitat. The cave is 50 m long, with two branches, acting as an intermittent overflow of the larger permanent spring Vrilo Rumin, situated 730 m east-southeast. Both springs are draining karstwater from middle part of Livansko Polje Basin (Bosnia and Herzegovina) Under Dinara Mts. towards upper Cetina River Valley. The following Hydrobiidae were detected in the locality: Horatia klecakiana, Orientalina curta germari, Kerkia jadertina sinjana (Kuščer, 1933) and a Montesieriid species of Paladilhiopsis Pavlović, 1913 and/or Lanzaia Brusina, 1906.

Discussion
All four valid species of Horatia described so far inhabit springs, but they have eyes and more or less pigmented soft parts, and can be classified as crenobiotic, at most as stygophiles (as defined by Pipan 2009, 2014). H. ozimeci sp. nov., with neither eyes nor any pigment, seems the first typically stygobiont Horatia. This single live specimen was most probably washed out from the cave together with few empty shells. Already Bourguignat (1887) noted the high variability of the shell of H. klecakiana (Fig. 1); later Radoman (1966Radoman ( , 1983 described and illustrated also the high variability of the penis in this species. The most characteristic feature of the shell of H. ozimeci is its partial scalarity -the open coil at the terminal part of the body whorl. There have been found also empty shells with the higher, conically elevated spire and more whorls visible within the umbilicus. Few empty shells found in the type locality were entirely scalariform, but they need not belong to H. ozimeci. Scalarity is characteristic for a few truncatelloidean species, e.g. Gocea ochridiana Hadžišče, 1956 from the Ochrid Lake (Radoman 1983), and several species from the Baikal Lake (Sitnikova et al. 2001;Clewing et al. 2015) but sometimes may be phenotypically determined (e.g. parasites, untypical chemistry: e.g. Fretter and Graham 1962), but is also typical feature of some species. In our case this morphological character is accompanied by molecular distinctness.
So far H. klecakiana was found only in the Cetina Valley and Livansko Polje Basin (Fig. 2). One of our sites was placed in the Ruda Valley and the second at Grab valley (both are left tributary of Cetina River); thus they are the first H. klecakiana localities outside the Cetina Valley and Livansko Polje.
High bootstrap support and p-distance between H. ozimeci and H. klecakiana confirm that they are two distinct species, belonging to the same genus Horatia. The evidence of species distinctness is especially strong since the two taxa occur in sympatry (inside the spring zone). The complete lack of polymorphism in the studied fragment of COI in the specimens of H. klecakiana from its five sequenced populations additionally strengthens the molecular difference between H. klecakiana and H. ozimeci as delimiting distinct species.
The second stygobiotic species H. stygorumina sp. nov. is known only as empty shells from cave sediments of its type locality, 17 km from the locality of H. ozimeci sp. nov. Despite the similarities in the shell morphology of both species, suggesting their congeneric position, the second species differs from H. ozimeci sp. nov. by more elevated conical spire and more close-set last whorl. The stygobiotic Horatia forms can be found in most of the large springs at left tributary of upper Cetina River (springs: Rumin, Kosinac, Gala, Beguša, Grab). It may suggest possibly a similar evolutionary adaptation as we can see in the geographically close H. ozimeci sp. nov.