Crenobiont, stygophile and stygobiont molluscs in the hydrographic area of the Trebišnjica River Basin

Abstract In the paper the crenobiont, stygophile and stygobiont malacofauna of the karst area of Popovo Polje around Trebinje (Eastern Herzegovina, BiH) is presented. The materials were collected from springs, caves and interstitial habitats (with a Bou-Rouch pump) at 23 localities. The following species were found: Pisidium cf. personatum A.W. Malm, 1855, Theodoxus callosus (Deshayes, 1833), Sadleriana fluminensis (Küster, 1852), Radomaniola curta (Küster, 1852), Radomaniola cf. bosniaca (Radoman, 1973), Kerkia briani Rysiewska & Osikowski, 2020, Montenegrospeum bogici (Pešić & Glöer, 2012), Litthabitella chilodia (Westerlund, 1886), Travunijana vruljakensis Grego & Glöer, 2019, a new genus and species of the Sadlerianinae, Emmericia ventricosa Brusina, 1870, Iglica cf. absoloni (A.J. Wagner, 1914), Plagigeyeria tribunicae Schütt, 1963, Paladilhiopsis arion Rysiewska & Osikowski, 2021, Valvata montenegrina Glöer & Pešić, 2008, Radix labiata (Rossmässler, 1835), Galba truncatula (O. F. Müller, 1774), Ancylus recurvus Martens, 1783, Ancylus sp. and the amphibiotic Succinea cf. putris (Linnaeus, 1758). The redescription of the genus Travunijana Grego & Glöer, 2019, applying the characteristics of shell, female reproductive organs and penis, is also presented. The new genus and species are described, based on the shell, penis, radula and fragmentary data on the female reproductive organs. For all species, the mitochondrial cytochrome oxidase subunit I (COI) is applied to confirm the determination; in the case of Travunijana and the new genus, the nuclear histone H3 locus is also used, in order to infer both their distinctiveness and phylogenetic relationships.


Introduction
The Dinaric Karst is a global hotspot for subterranean biodiversity. This is particularly true in the case of its stygobiont, stygophilic and crenobiont communities. The present paper focusses on providing further evidence of one generally under-reported aspect of freshwater aquatic biodiversity -namely the malacofauna of the Trebišnjica River Basin, predominantly in the hydrographically complex karst area of Eastern Herzegovina in Bosnia and Herzegovina.
The study reported below, was undertaken under the remit of the RS-Bosnia and Herzegovina Official Government Licence, which is granted annually to the "Proteus Project in Bosnia and Herzegovina" to undertake its objective of protecting and conserving endangered cave fauna and by extension, to protect and conservationmanage the natural karst conduit-aquifer hypogean ecosystems containing the endangered cave faunal species. One of the objectives of the Project is to fully characterise these ecosystems and in doing so, to provide an inventory of their biodiversity.
In this context, the contribution made by the visiting team of malacologists from the Department of Malacology of the Jagiellonian University's Institute of Zoology and Biomedical Research and from Department of Animal Reproduction, Anatomy and Genomics of University of Agriculture in Krakow, both in Poland, has provided the "Proteus Project" with vital information on the biological characteristics and geographic distribution of a range of genera and species of malacofauna collected at 23 locations connected to 11 separate karst conduit-aquifer ecosystems across a wide area of the Trebišnjica River Basin. The 23 sampling locations were purposely selected by the Director of the "Proteus Project" to represent a typical range of karst hydrological features, such as cave resurgence springs (vrelo), ponors and estavelles, either underground or at surface outlets or inlets.
Speleomalacological research on this scale and in such an integrated form, has never been undertaken before now in Bosnia and Herzegovina. Not surprisingly, therefore, the Polish team has identified a new genus and species of meiofaunal gastropod (Mollusca). As a standalone account, these first results, containing verifiable genomic data are of great scientific importance in their own right, but when combined with the associated variety of environmental data being amassed by the "Proteus Project", they assume a much greater value.
In regard to both ecosystem services and as a nutrient-rich food supply, the importance of the position of malacofauna near the bottom of the "foodchain" of the subterranean aquatic ecosystem, cannot be overstated. Without them being present in all their wonderful variety and population numbers, the diversity of many of the higher cave animals would certainly not be as great.

Material and methods
In June and September 2019, we collected aquatic gastropods from springs, interstitial habitats and caves at 23 localities (Table 1, Figs 1-3). They were either collected by hand and sieve in caves and springs, or with a pump applying the Bou-Rouch technique (Bou and Rouch 1967), to sample interstitial fauna below the sedimented floor Figure 1. Selected studied localities from Trebinje area, part 1 A locality 1, Vrelo "Vrijeka" (Bijeljani), Dabarsko Polje B locality 5, Vrelo "Pokrivenik" (Muhareva Ljut), Popovo Polje C locality 6, Vrelo "Lukavac" (Zavala) D locality 9, Izvor "Knez" (Trklja) E pumping of interstitial fauna at locality 11, Vrelo "Tučevac" (Mostaći) F locality 13, Vrelo "Polički Studenac" (Crkvina). See also Table 1. of streams, at a depth of about 50 cm. The tube was inserted in the sediment five times, and 20 litres were pumped each time. Samples were sieved through a 500 μm sieve and fixed in 80% analytically pure ethanol, replaced twice, and later sorted. Next, the snails were put in fresh 80% analytically pure ethanol and kept at -20 °C temperature in a refrigerator. Percentages of each identified taxon in each locality are presented in Table 1, with division into samples collected on the surface and with a pump.
The shells were photographed with a Canon EOS 50D digital camera, under a Nikon SMZ18 microscope. The dissections were done under a Nikon SMZ18 microscope with dark field, equipped with Nikon DS-5 digital camera, whose captured images were used to draw anatomical structures with a graphic tablet. Morphometric parameters of the shell were measured by one person using a Nikon DS-5 digital camera and ImageJ image analysis software (Rueden et al. 2017). The radulae were extracted with Clorox, applying the techniques described by Falniowski (1990), and examined and photographed using a HITACHI S-4700 scanning electron microscope.
Mitochondrial cytochrome oxidase subunit I (COI) and nuclear histone 3 (H3) loci were sequenced. Details of PCR conditions, primers used and sequencing technique were as given in Szarowska et al. (2016a). Sequences were initially aligned in the MUSCLE (Edgar 2004) programme in MEGA 7 (Kumar et al. 2016) and then checked in BIOED-IT 7.1.3.0 (Hall 1999). Uncorrected p-distances were calculated in MEGA 7. Estimation of the proportion of invariant sites and the saturation test (Xia 2000;Xia et al. 2003) were performed using DAMBE (Xia 2018). In the phylogenetic analysis, additional sequences from GenBank were used (Table 2). The phylogenetic analysis was performed applying two approaches: Bayesian Inference (BI) and Maximum Likelihood (ML). The Bayesian analyses were run using MrBayes v. 3.2.3 (Ronquist et al. 2012) with defaults for most priors. Two simultaneous analyses were performed, each with 10,000,000 generations,  This location is permanently hydrologically active and its water originates from ponors in Ljubomirsko Polje 14 km away. Although it is locally regarded as a vrelo, it is actually an estavelle. This was once used as a public water supply. Litthabitella chilodia 14.6/0 A permanently hydrologically active outlet from a complex karst conduit-aquifer, whose principal source is unproven. This was once a public water supply. Emmericia ventricosa 0/100 The entrance comprises a neo-circular stone wall leading down into the interior by more than 20 stone steps set into the natural stone floor of the karst conduit. The construction is of Austro-Hungarian origin and designed to give easy access to the potable water supply for local people. The location was hydrologically inactive when sampled. 23 River Konavoska Ljuta (Ljuta), Croatia; samples from the surface (Stones, plants) This karst river originates from Vrelo "Konavoska Ljuta" a few metres upstream from the sampling location. However, the water itself originates from a ponor 10 km away in Zubačko Polje near Trebinje in Eastern Herzegovina. This cave resurgence spring is used as a public water supply. The samples were collected under low-flow conditions.
Radomaniola curta 84.4/100 with one cold chain and three heated chains, starting from random trees and sampling the trees every 1000 generations. The first 25% of the trees were discarded as burn-in. The analyses were summarised as a 50% majority-rule tree. The Maximum Likelihood analysis was conducted in RAxML v. 8.2.12 (Stamatakis 2014) using the RAxML-HPC v.8 on XSEDE (8.2.12) tool via the CIPRES Science Gateway (Miller et al. 2010). We applied the GTR model whose parameters were estimated by RAxML (Stamatakis 2014).

Bivalvia Pisidiidae
Pisidium cf. personatum A.W. Malm, 1855 Remarks. Specimens of this common, widely distributed, Holarctic and eurybiotic species were found in many springs. It was also collected from interstitial habitats (with a Bou-Rouch pump) at the localities 12, 21 and 23 (Fig. 4). Remarks. This species, described from Greece and reported from Greece and Albania, was found at some larger springs, but never in subterranean waters.

Radomaniola Szarowska, 2006
Remarks. Replacement name for Orientalina Radoman, 1978. The genus is widely spread in the former Yugoslavia, but recorded also from Italy. Radoman (1983) distinguished six species of Radomaniola, and in one of them -R. curta -eight subspecies. It has to be noted that in modern phylogenetics, the only acceptable meaning of a subspecies is a geographic race, which was hardly the case in Radoman's classification; also, far from being acceptable is that all his species-level taxonomy was based on the shell alone, strikingly variable in this genus (e.g., ; see also   Table 1. species discrimination in Radomaniola should be studied with more extensive material, which we are proposing to do. At the moment, considering only Radomaniola from the area sampled in this study, one can distinguish two main clades (Fig. 6), representing at least two distinct species. For the one including the sequences of the snails from the spring at Vranjicke Njive, type locality of Radomaniola curta curta (sequences KC011781 and KC011784), we used a provisional assignment to this species; for the second clade we provisionally used the name R. cf. bosniaca. In general, the representatives of Radomaniola were the most common snails at the studied localities, and were found at the surface, as well as in the pumped interstitial samples and could also be found in caves. Radomaniola, pigmented and with eyes, is a stygophile gastropod.  Remarks. Collected at the localities 1, 6, 12, 13, 14 and 18 (Fig. 4) on the surface, but only at the localities 3 and 11 interstitially. At the locality 13 in sympatry with R. curta. Remarks. Found at the locality 13 (Fig. 4), its type locality, and at locality 17 (about 1 km away), where it is an element of the meiofauna; pumped with a Bou-Rouch pump (Hofman et al. 2020b).   collected live specimens, and described the lack of eyes and pigment and the penis with a lobe at its medial part. They considered B. bogici as belonging to a new genus: Montenegrospeum . Later, Falniowski et al. (2014 demonstrated with molecular data that Montenegrospeum belongs to the Hydrobiidae, not Moitessieriidae, despite striking similarity of the shell between this snail and e.g., Iglica Wagner, 1927. Numerous live specimens of this species were pumped from interstitial habitats at the localities 1, 7 and 21 (Fig. 4). Remarks. This species was found at the localities 7, 8, 9, 20 and 21 (Fig. 4). It was numerous and was also found in a cave and sometimes interstitially; pumped. Fig. 9 GenBank no. COI: MW879256-MW879272; H3: MW865737-MW865748

Travunijana vruljakensis Grego & Glöer, 2019
Remarks. Grego and Glöer (2019) described a new monotypic genus Travunijana from Vrelo "Goricki Studenac" (Gorica), a spring at the right bank of the Trebišnjica River, this being its type locality. They found it also in two other springs: Vrelo Vruljak 1 (Gorica; our locality 12), and Vrelo Vruljak 3 (Gorica). Their diagnosis of the genus was based on a single "unique" character -the strange morphology of the penis -which was based on artefactual appearance, caused by fixation: a nonglandular outgrowth on the left side, located distally . The penis photographed by them presents a bulbous, drastically contracted distal section, making copulation impossible.
Our molecular data (Fig. 8) confirmed the distinctiveness of the genus Travunijana . The phylograms based on H3, as well as on both concatenated loci placed Travunijana as the sister species with Graecoarganiella Falniowski &Sarajana Radoman, 1975 (bootstrap 85%). The shell habitus is different (conic in Travunijana, ovate-conic in Sarajana), and the penial morphology differs : the outgrowth on the left side is simple and filamentous in Sarajana, and short and bi-lobed in Travunijana. The phylogram based on COI showed a more complicated pattern, but bootstrap supports were too low for any more certain placement in the phylogeny.

Redescription of the genus Travunijana Grego & Glöer, 2019
Diagnosis. Shell conic with moderately convex whorls, big sphaerical bursa copulatrix and two nearly vestigial receptacula seminis, penis long and slender, distally forming a slightly bent filament, at the base of the filament an outgrowth on the left side of the penis, formed of two finger-like lobes.
Description. The shell (Fig. 9) as described by Grego and Glöer (2019). The female reproductive organs (Fig. 10) with bulbous loop of (renal) oviduct, big and spherical bursa copulatrix and two nearly vestigial receptacula seminis: proximal (rs 2 of Radoman 1973) and distal (rs 1 of Radoman 1973) one. The penis ( Fig. 11) long and slender, slightly bent at its medial section, at the base of the long filamentous distal section and an outgrowth on the left side, consisted of two finger-like lobes.

Type species. Iglicopsis butoti by original designation
Diagnosis. Shell ovate-conic with broad and flat apex, transparent, operculum smooth, no pigment, eyes absent, ctenidium present, penis long, tapering, with bilobed outgrowth on the left side and flat outgrowth at the right side, unpigmented renal oviduct, bursa copulatrix and two small receptacula seminis.
Remarks. Iglicopsis shell resembles that of Montenegrospeum, but is more oval, with broader spire and broader flat apex, sometimes showing scalarity at the body whorl; the penis with the left-side outgrowth located more proximally and bi-lobed and additional flat outgrowth on the right side; the molecular divergence (p = 0.186 for mitochondrial COI and p = 0.114 for nuclear H3) at the genus-level.  Table 3). Scale bars: 1 mm. Diagnosis. Shell minute, ovate-conic, distinguishable from Montenegrospeum by a more oval habitus, broader spire and broader flat apex, sometimes showing scalarity at the body whorl; the penis with the left-side outgrowth located more proximally and bi-lobed, and additional flat outgrowth on the right side. Description. Shell ( Fig. 9M-P) up to 1.49 mm high and 0.55 mm broad, ovateconic, whitish, translucent, thin-walled, and consisting of about five whorls, growing regularly and separated by moderately deep suture. Spire high and broad, apex broad and flat, body whorl less than 0.5 of the shell height, Aperture small, prosocline, oval in shape, peristome complete and thin, somewhat swollen, in contact with the wall of the body whorl, in some specimens showing scalarity close to the aperture, umbilicus slit-like. Shell surface smooth, with growth lines hardly visible.

Iglicopsis butoti
Measurements of holotype and sequenced and illustrated shells: Table 3. Shell variability slight; scalarity and much bigger dimensions of one specimen (Fig. 9P) most probably caused by the larval Trematoda (parasite gigantism).
Soft parts morphology and anatomy. Body white, pigmentless, with no eyes. Ctenidium with nine short lamellae, osphradium elongated. Tectum forming a characteristic broad loop (Fig. 9N). Female reproductive organs with unpigmented renal oviduct, bursa copulatrix and two small receptacula seminis; details unknown.
The radula (Fig. 12) with the central tooth cusp formula: (4)3 1 3(4) 1 1 − − − or (5)4 1 4(5) 1 1   Rather long and slender cusps grow regularly to central one. Lateral cusp with 5 -1 -5(6) long and massive cusps. Inner marginal tooth with ca 23 slender cusps of nearly invariable length along the tooth edge, outer marginal tooth with 26 broadly triangular cusps. Penis (Fig. 13A) long, tapering, below the half of its length, proximally, bi-lobed outgrowth on the left side and flat outgrowth at the right side, at the distal part and the vas deferens well visible inside, running in zigzags.

− − −
Derivatio nominis. The genus name refers to the similarity of the shell to the moitessieriid genus Iglica Wagner, 1927. The specific epithet butoti refers to the memory of Dr Louis J. M. Butot, a Dutch malacologist devoted mostly to the Greek malacofauna, good friend and the mentor of AF.
Distribution and habitat. Known from the type locality only. Molecular relationships. despite its shell morphology, Iglicopsis clearly belongs to the Hydrobiidae Stimpson, 1865, Sadlerianinae Szarowska, 2006, and not to the Moitessieriidae Bourguignat, 1863 (Fig. 8). Its sister species is Montenegrospeum bogici in the H3 tree (Fig. 8, bootstrap 95%), and on the tree based on both concatenated loci (but with bootstrap 63% only); in the COI tree the bootstrap does not support its phylogenetic position. Remarks. The species was found at the localities 1, 5, 12, 13, 22 (estavelle) (Fig.  4), at the surface. Molecular data rather confirms its distinctiveness (p = 0.038) from E. expansilabris (Bourguignat, 1870), described from Vrelo "Ombla" on the Dalmatian coast in nearby Croatia.

Iglica cf. absoloni (A.J. Wagner, 1914)
Remark. Empty shell was found interstitially at the locality 20 (Fig. 4). Schütt, 1963 Remark. Empty and incomplete shell was found interstitially at the locality 17 (Fig. 4). Remarks. Live specimens were pumped from an interstitial habitat at the locality 21 (Fig. 4). They were recently described as new to science . Morphologically and molecularly, they were distinct from the moitessieriid species discussed in Hofman et al. (2018). Rysiewska et al. (2021) demonstrated that at least some of the species assigned to the genus Plagigeyeria Tomlin, 1930 belong to the genus Paladilhiopsis Pavlović, 1913. Our specimens from Gučina in Trebinje molecularly formed the sister clade with Plagigeyeria montenegrina Bole, 1961 from Obodska Pečina in Montenegro. Also, the outline and orientation of the long axis of the aperture was characteristic of Plagigeyeria. The similarly shaped shell and geographic range may suggest assignment to P. nitida Schütt, 1963, but the number of whorls of our specimens is much higher than presented by Schütt (1972). Fig. 14F GenBank no. COI: MZ027632-MZ027633

Pulmonata Lymnaeidae
Radix labiata (Rossmässler, 1835) Fig. 14G GenBank no. COI: MZ027630 Remarks. This common Central-European and Mediterranean species was found at the localities 16 and 20 (Fig. 4). Inhabits slowly running or stagnant small water bodies (e.g., Glöer 2019), preferably close to ground waters, but not found in subterranean habitats. Remarks. Common Palaearctic gastropod, inhabiting nearly all of Europe. This amphibious and calcifilous (e.g., Glöer 2019) species inhabits small water bodies, rich in vegetation, such as at our locality 16 -a small lake in a collapsed cave, rather than subterranean habitats, but at the locality 15 it was found in an estavelle, a kind of vast subterranean tunnel transporting water either down, as outlet of surface waters, or up, forming temporary active springs. Shells of our specimens (Fig. 14H, I) were somewhat untypical, with low and broad spire, but the variation of the shell in the Lymnaeidae has been long known (e.g., Roszkowski 1914;Falniowski 1980Falniowski , 1981, as being wider than in any other gastropod group. Remarks. Ancylus is known as a stygophile gastropod (e.g., Culver and Pipan 2009;Macher et al. 2016;personal observations); also inhabiting caves. Ancylus recurvus at the locality 13 was also found interstitially, pumped, and at the locality 15 (Fig. 4) it inhabited an estavelle. Our A. recurvus molecularly belonged to the clade "Ancylus sp. B" of Albrecht et al. (2006), Clade 3 of Pfenninger et al. (2003) (Fig. 15). It is molecularly different from A. fluviatilis by 9%.

Ancylus recurvus Martens, 1783
Ancylus sp. Remarks. Considering the shell morphology, it should be determined as A. fluviatilis O. F. Müller, 1774, a species reported from this region. However, Pfenninger et al. (2003) demonstrated that A. fluviatilis inhabits a wide range throughout Europe, but in the southern regions there are a few cryptic, molecularly defined species of Ancylus. Our Ancylus sp. molecularly belonged to the Clade 4 of Pfenninger et al. (2003) and "Ancylus sp. C4" of Albrecht et al. (2006) (Fig. 15). It was found as a crenobiont in the cave springs at the localities 8, 9 and 16 (Fig. 4). Molecular divergence between this Ancylus sp. and Ancylus recurvus is 7%, and similar value (7.5%) is observed between this Ancylus sp. and A. fluviatilis.

Stylommatophora: Succineidae
Succinea cf. putris (Linnaeus, 1758) Fig. 14N GenBank no. COI: MZ027631 Remarks. Our specimen differed by 12 substitutions (97.55% of identity) from Succinea sp. GenBank number KF412772 from "Egypt: Fayoum Governorate". For the closest European Succinea, S. putris the identity was only 86.73%. In fact, this value is close to the threshold one to distinguish species in the Pulmonata, thus our specimen may represent some still unsequenced species of Succinea. This amphibious snail was found at locality 10 (Fig. 4).