A peer-reviewed open-access journal ZooKeys 1047: 61–89 (2021) doi: 10.3897/zookeys.1047.64034 https://zookeys.pensoft.net Crenobiont, stygophile and stygobiont molluscs in the hydrographic area of the Trebišnjica River Basin Andrzej Falniowski1, Brian Lewarne2, Aleksandra Rysiewska1 , Artur Osikowski3, Sebastian Hofman4 1 Department of Malacology, Institute of Zoology and Biomedical Research, Jagiellonian University, ul. Gron­ostajowa 9, 30-387 Kraków, Poland 2 The Devon Karst Research Society, Library & Office, 46, Morley Court, Western Approach, Plymouth, Devon, UK 3 Department of Animal Reproduction, Anatomy and Genomics, University of Agriculture in Krakow, al. Mickiewicza 24/28, 30-059 Kraków, Poland 4 Department of Com­parative Anatomy, Institute of Zoology and Biomedical Research, Jagiellonian University, ul. Gronostajowa 9, 30-387 Kraków, Poland Corresponding author: Artur Osikowski (a.osikowski@urk.edu.pl) Academic editor: E. Gittenberger | Received 5 February 2021 | Accepted 24 April 2021 | Published 28 June 2021 http://zoobank.org/835A7E2B-82E8-45B0-9E3F-B8548031A048 Citation: Falniowski A, Lewarne B, Rysiewska A, Osikowski A, Hofman S (2021) Crenobiont, stygophile and stygobiont molluscs in the hydrographic area of the Trebišnjica River Basin. ZooKeys 1047: 61–89. https://doi. org/10.3897/zookeys.1047.64034 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. Copyright Andrzej Falniowski et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Keywords Balkans, Bosnia and Herzegovina, cave, COI, H3, karst area, meiofauna, molecular systematics, new genus, new species, spring 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. 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. 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 2. Selected studied localities from Trebinje area, part 2 A locality 14, Vrelo “Oko” (Zasad) B loca-lity 16, Igorovo Jezero (lake) (Gorica) C locality 17, Vrelo „Vruljak 2” (Gorica), Trebinjsko Polje D local­ity 20, confluence of Sušica River and Jazina River (Jazina). 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. DNA was extracted from whole specimens; tissues were hydrated in 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 tris-EDTA (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) 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 BIOED­IT 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, Table 1. The list of studied localities, with a short description of their characteristics, geographical coor­dinates and taxa identified. Id Site names, characteristics and codes Coordinates Taxa confirmed % of taxa in site (surface/ pump) 1 Vrelo „Vrijeka” (Bijeljani), Dabarsko Polje; at the outlet (BiH19_08) A permanent cave resurgence spring whose water originates from ponors located in Lukavačko Polje. 43.07417, 18.23899 Emmericia ventricosa Montenegrospeum bogici Radomaniola cf. bosniaca 0/12.6 100/0 0/87.4 2 Estavela „Ljelješnica”(Bijeljani); inside the cave (BiH19_14) When checked, this location was hydrologically inactive. 43.05400, 18.24069 – – 3 Rijeka (river) „Vrijeka” (Dabarsko Polje); on the surface near entrance of Ponor „Ponikva” (BiH19_15) Samples taken under low-flow conditions. 43.04535, 18.25217 Radomaniola cf. bosniaca 100/0 4 Estavela „Kapuša” (Dračevo); inside the entrance (BiH19_24) Checked when the estavelle was hydrologically inactive. 42.85692, 18.07665 – – 5 Vrelo „Pokrivenik” (Muhareva Ljut), Popovo Polje; spring at the cave entrance; high water level variation (BiH19_05) Samples taken when the location was hydrologically inactive. 42.85166, 17.99838 Emmericia ventricosa 0/100 6 Vrelo „Lukavac” (Zavala); outlet for Vjetrenica Pećina. Spring below the cave entrance; high water level variation (BiH19_06) Samples taken when the location was hydrologically inactive. 42.84643, 17.9846 Radomaniola cf. bosniaca 0/100 7 Vrelo „Bitomišlje” (Golubinac); in valley above Zavala, with Austro-Hungarian infrastructure (BiH19_07) Samples taken under extremely low-flow conditions. 42.83799, 17.97161 Litthabitella chilodia Montenegrospeum bogici 40.3/0 59.7/0 8 Izvor „Kneginja” (Trklja); a low-flow groundwater spring in Dolomite coming from a limestone blockhouse (BiH19_20) 42.75729, 18.3693 Ancylus sp. Litthabitella chilodia 0/2.7 0/97.3 9 Izvor „Knez” (Trklja); a low-flow groundwater spring in Dolomite coming from a limestone blockhouse (BiH19_21) 42.75463, 18.37218 Ancylus sp. Litthabitella chilodia 0/2.3 0/97.7 10 Confluence of Trebišnjica River with the Potok (stream) Blace (Blace); surface stream from a cave spring-group on the right bank of Trebišnjica River (BiH19_17) 42.71536, 18.35077 Radomaniola curta Sadleriana fluminensis Succinea cf. putris 100/32.1 0/64.3 0/2.6 11 Vrelo „Tučevac” (Mostaći); the spring inside the cave (BiH19_13) A high-level overflow spring from a locally complex estavelle cave system. When active, its water originates from ponors in Ljubomirsko Polje 14 km away. This was hydrologically inactive when sampled. 42.71445, 18.30278 Radomaniola cf. bosniaca 100/0 12 Vrelo „Vruljak 1” (Gorica), Trebinjsko Polje. This was sampled in the resurgence pool before which 2 cave rivers Rijeka “Goričica” and Rijeka “Vrulje” have joined inside & emerge (BiH19_03) The cave resurgence spring is just one outlet from a locally very complex cave system, containing a very rich biodiversity. The water originates from ponors in Ljubomirsko Polje about 12 km away. 42.71393, 18.36833 Emmericia ventricosa Pisidium cf. personatum Radomaniola cf. bosniaca Travunijana vruljakensis 0/7.8 50/0 0/92.2 50/0 13 Vrelo „Polički Studenac” (Crkvina); a cave spring in the left bank of Trebišnjica River (BiH19_11) 42.71288, 18.36514 Ancylus recurvus Emmericia ventricosa Iglicopsis butoti sp. nov. Kerkia briani Radomaniola curta Radomaniola cf. bosniaca Travunijana vruljakensis 3.7/0 0/44.3 27.8/0 38.9/0 10.2/7.6 0/48.1 19.4/0 14 Vrelo “Oko” (Zasad); a spring in the entrance to the cave system; surrounded by ancient limestone-block housing; at the centre of Trebinje (BiH19_23) 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. 42.71274, 18.33697 Radomaniola cf. bosniaca Travunijana vruljakensis 0/5.9 0/94.1 15 Estavela „Pećine” (Mostaći) (BiH19_12) This is a major estavelle-type outlet for the karst conduit-aquifer originating at the ponors in Ljubomirsko Polje. It was hydrologically inactive when sampled. 42.71244, 18.30497 Ancylus recurvus Galba truncatula 100/0 0/100 16 Igorovo Jezero (lake) (Gorica); small lake in a collapsed cave passage with cave springs and containing many ponors; muddy bottom (BiH19_19) The water originates from ponors in both Ljubomirsko Polje and Cibrijansko Polje. The ponors in and around the lake feed water underground downstream to Vrelo “Vruljak 2” (Gorica). 42.71111, 18.38495 Ancylus sp. Galba truncatula Radix labiata Sadleriana fluminensis 0/9.1 0/36.4 0/9.1 0/45.4 Id Site names, characteristics and codes Coordinates Taxa confirmed % of taxa in site (surface/ pump) 17 Vrelo „Vruljak 2” (Gorica), Trebinjsko Polje; this location was sampled at the resurgence spring outlet before which 2 cave rivers have joined inside: Rijeka “Pešterčica” and Rijeka “Venator” (BiH19_02) This is a permanently hydrologically active outlet from a locally very complex cave system containing a very rich biodiversity. 42.71062, 18.37618 Kerkia briani Plagigeyeria tribunicae Radomaniola curta Sadleriana fluminensis Travunijana vruljakensis 15.9/0 2.3/0 0/96.5 0/3.5 81.8/0 18 The intermittently active cave spring, Vrelo „Vražiji Mlin” (D. Grančarevo); Trebišnjica Canyon (BiH19_04) This is fed by ponors in Jasen Polje. The location is set in dolomitic limestone. 42.70847, 18.44801 Radomaniola cf. bosniaca 0/100 19 “Slomljen pecina” (Mokri Dolovi); (BiH19_22) Since being sampled, this location has now been buried and made inaccessible by urban development. 42.70844, 18.35419 – – 20 Confluence of Sušica River and Jazina River (Jazina) (BiH19_16) This was sampled under low-flow conditions. The source of the water is a giant estavelle situated in karstified dolomite with dolomitic limestone. 42.70429, 18.50491 Iglica cf. absoloni Litthabitella chilodia Radix labiata Valvata montenegrina 16.7/0 83.3/0 0/72.2 0/27.8 21 Vrelo „Lušac” (Gučina); at the surface outlet (BiH19_10) A permanently hydrologically active outlet from a complex karst conduit-aquifer, whose principal source is unproven. This was once a public water supply. 42.70111, 18.3575 Litthabitella chilodia Montenegrospeum bogici Pisidium cf. personatum Paladilhiopsis arion Travunijana vruljakensis 14.6/0 22.0/0 4.9/0 58.5/0 0/100 22 Estavela „Mali Šumet” (Bugovina), Mokro Polje: in the entrance shaft (BiH19_01) 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. 42.65665, 18.34458 Emmericia ventricosa 0/100 23 River Konavoska Ljuta (Ljuta), Croatia; samples from the surface (Stones, plants) (BiH19_18) 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. 42.53408, 18.37647 Pisidium cf. personatum Radomaniola curta 15.6/0 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). Systematic part 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). Table 2. Taxa used for phylogenetic analyses with their GenBank accession numbers and references. Species COI/H3 GB numbers References Agrafia wiktori Szarowska & Falniowski, 2011 JF906762/MG543158 Szarowska and Falniowski 2011/Grego et al. 2017 Alzoniella finalina Giusti & Bodon, 1984 AF367650/-Wilke et al. 2001 Anagastina zetavalis (Radoman, 1973) EF070616/-Szarowska 2006 Ancylus sp. B DQ301830 DQ301838/-Albrecht et al. 2006 Ancylus sp. C4 KY012232 KY012163/-Macher et al. 2016 Ancylus sp. – clade 3 AY350516 AY350519/-Pfenninger et al. 2003 Ancylus sp. – clade 4 AY350520 AY350521/-Pfenninger et al. 2003 Avenionia brevis berenguieri (Bourguignat, 1882) AF367638/-Wilke et al. 2001 Belgrandia thermalis (Linnaeus, 1767) AF367648/-Wilke et al. 2001 Belgrandiella kuesteri (Boeters, 1970) MG551325/-Osikowski et al. 2018 Belgrandiella kusceri (A. J. Wagner, 1914) -/MG551366 Osikowski et al. 2018 Bithynia tentaculata (Linnaeus, 1758) AF367643/-Wilke et al. 2001 Bracenica gloeri Grego, Fehér & Erőss, 2020 MT396209/-Hofman et al. 2020a Bythinella cretensis Schütt, 1980 KT353689/-Szarowska et al. 2016b Bythiospeum acicula (Hartmann, 1821) KU341350/MK609536 Richling et al. 2016/Falniowski et al. 2019 Daphniola louisi Falniowski & Szarowska, 2000 KM887915/-Szarowska et al. 2014a Dalmatinella fluviatilis Radoman, 1973 KC344541/-Falniowski and Szarowska 2013 Dalmatinella simonae Beran & Rysiewska, 2021 MT773271/-Beran et al. 2021 Ecrobia maritima (Milaschewitsch, 1916) KX355835/MG551322 Osikowski et al. 2016/Grego et al. 2017 Emmericia expansilabris Bourguignat, 1880 KC810060/-Szarowska and Falniowski 2013a Fissuria boui Boeters, 1981 AF367654/-Wilke et al. 2001 Graecoarganiella parnassiana Falniowski & Szarowska, 2011 JN202352/-Falniowski and Szarowska 2011 Graecoarganiella sp. JN202353/MN03140 Falniowski and Szarowska 2011/Hofman et al. 2019 Graziana alpestris (Frauenfeld, 1863) AF367641/-Wilke et al. 2001 Grossuana hohenackeri (Küster, 1853) KC011749/-Falniowski et al. 2012 Hauffenia michleri (Kuščer, 1932) KT236156/KY087878 Falniowski and Szarowska 2015 /Rysiewska et al. 2017 Heleobia maltzani (Westerlund, 1886) KM213723/MK609534 Szarowska et al. 2014b/ Falniowski et al. 2019 Horatia klecakiana Bourguignat, 1887 KJ159128/-Szarowska and Falniowski 2014 Iglica gracilis (Clessin, 1882) MH720985/MH721003 Hofman et al. 2018 Islamia zermanica (Radoman, 1973) KU662362/MG551320 Beran et al. 2016/Grego et al. 2017 Littorina littorea (Linnaeus, 1758) KF644330/KP113574 Layton et al. 2014/unpub. Lithoglyphus prasinus (Küster, 1852) JX073651/-Falniowski and Szarowska 2012 Marstoniopsis insubrica (Küster, 1853) AF322408/-Falniowski and Wilke 2001 Moitessieria cf. puteana Coutagne, 1883 AF367635/MH721012 Wilke et al. 2001/Hofman et al. 2018 Montenegrospeum bogici (Pešić & Glöer, 2012) KM875510/MG880218 Falniowski et al. 2014/Grego et al. 2018 Montenegrospeum sketi Grego & Glöer, 2018 MG880216/-Grego et al. 2018 Paladilhiopsis grobbeni Kuščer, 1928 MH720991/MH721014 Hofman et al. 2018 Pontobelgrandiella sp. Radoman, 1978 KU497024/MG551321 Rysiewska et al. 2016/Grego et al. 2017 Pseudamnicola pieperi (Schütt, 1980) KT710670/-Szarowska et al. 2016a Pseudorientalia sp. KJ920477/-Szarowska et al. 2014c Radomaniola curta (Küster, 1853) KC011814/-Falniowski et al. 2012 Radomaniola curta curta (Küster, 1853) KC011781 KC011784 Falniowski et al. 2012 KC011787 KC011788 KC011791 KC011792 KC011810/- Radomaniola sp. KC011727 KC011745 Falniowski et al. 2012 KC011747 KC011763 KC011764 KC011766/-Sadleriana fluminensis (Küster, 1853) KF193067/-Szarowska and Falniowski 2013b Sarajana apfelbecki (Brancsik, 1888) MN031432/MN031438 Hofman et al. 2019 Sarajana cf. apfelbecki MN031431/-Hofman et al. 2019 Tanousia zrmanjae (Brusina, 1866) KU041812/-Beran et al. 2015 Gastropoda Neritopsina: Neritidae Theodoxus callosus (Deshayes, 1833) Remarks. This species, described from Greece and reported from Greece and Albania, was found at some larger springs, but never in subterranean waters. Figure 4. Distribution of the studied taxa. Localities' numbers correspond to Table 1. Caenogastropoda Hydrobiidae: Sadlerianinae Sadleriana fluminensis (Küster, 1852) Fig. 5A GenBank no. COI: MZ027620–MZ027622 Remarks. The most widely distributed species of Sadleriana. Found at the locali­ties 10, 16 and 17 (Fig. 4). 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., Falniowski et al. 2012; see also Fig. 5B–M). Molecular and anatomical data (Falniowski et al. 2012) did not confirm the classification of Radoman (1983), but demonstrated high genetic diversity, sug­gesting a flock of distinct species. The phylogeography as well as molecularly-based Figure 5. Shells of the studied gastropods: A Sadleriana fluminensis, locality 10 B–M Radomaniola B–H R. curta (localities: B–D – 10, E, F – 13, G – 17, H – 23) I–M R. cf. bosniaca (localities: I–K – 1, L, M – 12). Scale bar: 1 mm. 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. Figure 6. Maximum likelihood (ML) phylogram of the studied Radomaniola, based on the partial cy­tochrome oxidase subunit I (COI) sequences, bootstrap supports given if >60%, together with Bayesian probabilities; topotypes of R. curta curta marked with asterisks. Radomaniola curta (Küster, 1852) Fig. 5B–H GenBank no. COI: MW879241–MW879250 Remarks. Found at the localities 10, 13 and 23 (Fig. 4) on the surface and also interstitially and at the locality 17 only on the surface. At the locality 13 in the spring Polički Studenac, in sympatry with R. cf. bosniaca. Radomaniola cf. bosniaca (Radoman, 1973) Fig. 5I–M GenBank no. COI: MW879222–MW879240 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. Kerkia briani Rysiewska & Osikowski, 2020 Fig. 7A–C GenBank no. COI: MT780191–MT780196; H3: MT786730–MT786735; Hof­man et al. 2020b 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). Montenegrospeum bogici (Pešić & Glöer, 2012) Fig. 7D–K GenBank no. COI: MZ266648–MZ266650 Remarks. Pešić and Glöer (2012) described a new species of Bythiospeum Bour­guignat, 1882: B. bogici Pešić & Glöer, 2012 from underground waters of Vrelo “Taban”, in central Montenegro. Their description was based on empty shells. Later they (Pešić and Glöer 2013) 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 Pešić & Glöer, 2013. 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). Figure 7. Shells of the studied gastropods: A–C Kerkia briani D–K Montenegrospeum bogici (localities: D–F – 1, G, H – 7, I – 13, J – 14, K – 21) L–O Litthabitella chilodia (localities: L, M – 17, N–O – 8). Scale bars: 1 mm. Litthabitella chilodia (Westerlund, 1886) Fig. 7L–O GenBank no. H3: MZ285059–MZ285063 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. Travunijana vruljakensis Grego & Glöer, 2019 Fig. 9 GenBank no. COI: MW879256–MW879272; H3: MW865737–MW865748 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 (Grego and Glöer 2019). 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 Grego & Glöer, 2019. The phylograms based on H3, as well as on both concat­enated loci placed Travunijana as the sister species with Graecoarganiella Falniowski & Szarowska, 2011, and Sarajana Radoman, 1975 (bootstrap 85%). The shell habitus is different (conic in Travunijana, ovate-conic in Sarajana), and the penial morphology differs (Hofman et al. 2019): 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 (rs2 of Radoman 1973) and distal (rs1 of Radoman 1973) one. The penis Figure 8. Phylogenetic relationships of Travunijana and Iglicopsis based on COI, H3 and concatenated loci; bootstrap supports given if over 60%, their values together with Bayesian probabilities. (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. Travunijana vruljakensis was common at the studied territory, found at the locali­ties 12, 13, 14, 17 and 21. At 12, 13 and 17 (Fig. 4) interstitially pumped. Iglicopsis Falniowski & Hofman, gen. nov. http://zoobank.org/77758877-EEF4-448E-B727-D5632F9E5F51 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 Figure 9. Shells: A–L Travunijana vruljakensis M–P Iglicopsis butoti M holotype N 2F61 O 2F68 P 2F69 (extraction numbers, see Table 3). Scale bars: 1 mm. 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. Iglicopsis butoti Falniowski & Hofman, sp. nov. http://zoobank.org/C1A9D0B0-4B10-4977-B69B-7C4C42BB19D3 Fig. 9M–P GenBank no. COI: MW879273–MW879275; H3: MW865749–MW865751 Type materials. Holotype. Ethanol-fixed specimen (Fig. 9M), Vrelo „Polički Stu­denac” (Crkvina); a cave spring in the left bank of and adjacent to the Trebišnjica River (N 42.71288, E 18.36514) (our locality 13, Fig. 4) close to Trebinje (Bosnia and Herzegovina), interstitially, 50 cm below the gravel floor of the spring; in the col­lection of the Department of Malacology of Jagiellonian University, voucher number ZMUJ-M.2651. Paratypes. Three paratypes destroyed to extract DNA, one specimen ethanol­fixed, in the collection of the Department of Malacology of Jagiellonian University, ZMUJ-M.2652. 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. Table 3. Shell measurements (in mm) of holotype and sequenced and illustrated specimens of Iglicopsis butoti sp. nov. For explanation of the symbols a-ß, see Fig. 13B. Holotype 2F61 2F68 2F69 a 1.49 1.29 1.35 1.87 b 0.55 0.54 0.54 0.70 c 0.43 0.39 0.43 0.44 d 0.80 0.62 0.67 0.93 e 0.37 0.34 0.35 0.44 . 90 89 90 90 ß 20 18 20 18 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. 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) -- (5)4 1 4(5) -- or 11 -11 - 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. Emmericiidae Emmericia ventricosa Brusina, 1870 Fig. 14A–C GenBank no. COI: MZ027623–MZ027627 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. Moitessieriidae Iglica cf. absoloni (A.J. Wagner, 1914) Remark. Empty shell was found interstitially at the locality 20 (Fig. 4). Plagigeyeria tribunicae Schütt, 1963 Remark. Empty and incomplete shell was found interstitially at the locality 17 (Fig. 4). Paladilhiopsis arion Rysiewska & Osikowski, 2021 Fig. 14D, E GenBank no. COI: MW741739–MW741740; H3: MW776424–MW776425 Remarks. Live specimens were pumped from an interstitial habitat at the local­ity 21 (Fig. 4). They were recently described as new to science (Hofman et al. 2021). 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). Heterobranchia Heterostropha: Valvatidae Valvata montenegrina Glöer & Pešić, 2008 Fig. 14F GenBank no. COI: MZ027632–MZ027633 Remark. Some specimens found at the locality 20 (Fig. 4); in the surface waters. 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. Galba truncatula (O. F. Müller, 1774) Fig. 14H, I GenBank no. COI: MZ027628–MZ027629 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 1980, 1981), as being wider than in any other gastropod group. Ancylidae Ancylus recurvus Martens, 1783 Fig. 14J, K GenBank no. COI: MW879251–MW879253 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 sp. Fig. 14L, M GenBank no. COI: MW879254–MW879255 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). Acknowledgements The study was supported by a grant from the National Science Centre 2017/25/B/ NZ8/01372 to Andrzej Falniowski. 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