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

Andrzej Falniowski', Brian Lewarne’, Aleksandra Rysiewska', Artur Osikowski?, Sebastian Hofman‘

| Department of Malacology, Institute of Zoology and Biomedical Research, Jagiellonian University, ul. Gron- ostajowa 9, 30-387 Krakéw, 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 Krakéw, Poland 4 Department of Com- parative Anatomy, Institute of Zoology and Biomedical Research, Jagiellonian University, ul. Gronostajowa 9, 30-387 Krakéw, 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://z00bank.org/835A7E2B-82E8-45 BO-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 Trebignjica 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 in- terstitial 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 (Kiister, 1852), Radomaniola curta (Kiister, 1852), Radomaniola cf. bosniaca (Radoman, 1973), Kerkia briani Rysiewska & Osikowski, 2020, Montenegrospeum bogici (Pesié & Gloer, 2012), Litthabitella chilodia (Westerlund, 1886), Travunijana vruljakensis Grego & Gléer, 2019, a new genus and species of the Sadlerianinae, Emmericia ventricosa Brusina, 1870, Iglica cf. absoloni (A.J. Wagner, 1914), Plagigeyeria tribunicae Schitt, 1963, Pal- adilhiopsis arion Rysiewska & Osikowski, 2021, Valvata montenegrina Gloer & PeSi¢, 2008, Radix labiata (Rossmiassler, 1835), Galba truncatula (O. FE. Miller, 1774), Ancylus recurvus Martens, 1783, Ancylus sp. and the amphibiotic Succinea cf. putris (Linnaeus, 1758). The redescription of the genus Travunijana Grego & Gléer, 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 fe- male 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.

62 Andrzej Falniowski et al. / ZooKeys 1047: 61-89 (2021)

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 Trebisnjica River Ba- sin, 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 “Pro- teus Project in Bosnia and Herzegovina” to undertake its objective of protecting and conserving endangered cave fauna and by extension, to protect and conservation- manage the natural karst conduit-aquifer hypogean ecosystems containing the en- dangered cave faunal species. One of the objectives of the Project is to fully charac- terise 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 geo- graphic 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 Trebisnjica 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 under- ground 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, there- fore, 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 im- portance 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.

Aquatic molluscs of the Trebi8njica River Basin 63

| liaise Pa 80 eee Figure |. 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 ,,Lu- kavac” (Zavala) D locality 9, Izvor ,,.Knez” (Trklja) E pumping of interstitial fauna at locality 11, Vrelo » Lucevac” (Mosta¢i) F locality 13, Vrelo ,,Poli¢ki 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 tech- nique (Bou and Rouch 1967), to sample interstitial fauna below the sedimented floor

64 Andrzej Falniowski et al. / ZooKeys 1047: 61-89 (2021)

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 local-

ity 20, confluence of SuSica 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 um 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 micro- scope with dark field, equipped with Nikon DS-5 digital camera, whose captured im- ages were used to draw anatomical structures with a graphic tablet. Morphometric pa- rameters of the shell were measured by one person using a Nikon DS-5 digital camera and Image] 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 x 10 min); then total genomic DNA was extracted with the SHERLOCK extraction kit (A&A Biotechnology), and the final product was dissolved in 20 pl of tris- EDTA (TE)

Aquatic molluscs of the Trebisnjica River Basin 65

Figure 3. Studied localities.

buffer. The extracted DNA was stored at -80 °C at the Department of Malacology, Insti- tute of Zoology and Biomedical Research, Jagiellonian University in Krakéw (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 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. Iwo simultaneous analyses were performed, each with 10,000,000 generations,

66 Andrzej Falniowski et al. / ZooKeys 1047: 61-89 (2021)

Table |. The list of studied localities, with a short description of their characteristics, geographical coor- dinates and taxa identified.

Site names, characteristics and codes Coordinates Taxa confirmed % of taxa in site (surface/ pump) Vrelo ,,Vrijeka” (Bijeljani), Dabarsko Polje; at the outlet (BiH19_08) 43.07417, | Emmericia ventricosa 0/12.6 A permanent cave resurgence spring whose water originates from ponors located in 18.23899 | Montenegrospeum bogici 100/0 Lukavacko Polje. Radomaniola cE. 0/87.4 bosniaca Estavela ,,Ljelje3nica” (Bijeljani); inside the cave (BiH19_14) 43.05400, - - When checked, this location was hydrologically inactive. 18.24069 Rijeka (river) ,,Vrijeka” (Dabarsko Polje); on the surface near entrance of Ponor 43.04535, Radomaniola cf. 100/0 »Ponikva’” (BiH19_15) 18.25217 bosniaca Samples taken under low-flow conditions. Estavela ,,Kapusa” (Dragevo); inside the entrance (BiH19_24) 42.85692, = — Checked when the estavelle was hydrologically inactive. 18.07665 Vrelo ,,Pokrivenik” (Muhareva Ljut), Popovo Polje; spring at the cave entrance; 42.85166, | LEmmericia ventricosa 0/100 high water level variation (BiH19_05) 17.99838 Samples taken when the location was hydrologically inactive. Vrelo ,,Lukavac” (Zavala); outlet for Vjetrenica Pecina. Spring below the cave 42.84643, Radomaniola cf. 0/100 entrance; high water level variation (BiH19_06) 17.9846 bosniaca

Samples taken when the location was hydrologically inactive. Vrelo ,,Bitomislje” (Golubinac); in valley above Zavala, with Austro-Hungarian 42.83799, Litthabitella chilodia 40.3/0 infrastructure (BiH19_07) 17.97161 Samples taken under extremely low-flow conditions. Montenegrospeum bogici| 59.710

Izvor ,,Kneginja” (Trklja); a low-flow groundwater spring in Dolomite coming 42.75729, Ancylus sp. 0/2.7 from a limestone blockhouse (BiH19_20) 18.3693 Litthabitella chilodia 0/97.3 Izvor ,,Knez” (Trklja); a low-flow groundwater spring in Dolomite coming from a 42.75463, Ancylus sp. 0/2.3 limestone blockhouse (BiH19_21) 18.37218 Litthabitella chilodia 0/97.7 Confluence of Trebignjica River with the Potok (stream) Blace (Blace); surface 42.71536, Radomaniola curta 100/32.1 stream from a cave spring-group on the right bank of Trebi8njica River (BiH19_17) 18.35077 | Sadleriana fluminensis 0/64.3

Succinea cf. putris 0/2.6 Vrelo ,,Tuéevac” (Mostaci); the spring inside the cave (BiH19_13) 42.71445, Radomaniola cf. 100/0 A high-level overflow spring from a locally complex estavelle cave system. When 18.30278 bosniaca active, its water originates from ponors in Ljubomirsko Polje 14 km away. This was hydrologically inactive when sampled. Vrelo ,,Vruljak 1” (Gorica), Trebinjsko Polje. This was sampled in the resurgence 42.71393, | Emmericia ventricosa 0/7.8 pool before which 2 cave rivers Rijeka “Goricica” and Rijeka “Vrulje” have joined 18.36833 | Pisidium cf. personatum 50/0 inside & emerge (BiH19_03) ‘The cave resurgence spring is just one outlet from a locally very complex cave Radomaniola cE. 0/92.2 system, containing a very rich biodiversity. The water originates from ponors in bosniaca Ljubomirsko Polje about 12 km away. Travunijana 50/0 vruljakensis Vrelo ,,Poli¢ki Studenac” (Crkvina); a cave spring in the left bank of TrebiSnjica 42.71288, Ancylus recurvus 3.7/0 River (BiH19_11) 18.36514 | Emmericia ventricosa 0/44.3 Iglicopsis butoti sp. nov. 27.8/0 Kerkia briani 38.9/0 Radomaniola curta 10.2/7.6 Radomaniola cf. 0/48.1 bosniaca Travunijana 19.4/0 vruljakensis Vrelo “Oko” (Zasad); a spring in the entrance to the cave system; surrounded by 42.71274, Radomaniola cf. 0/5.9 ancient limestone-block housing; at the centre of Trebinje (BiH19_23) 18.33697 bosniaca This location is permanently hydrologically active and its water originates from Travunijana 0/94.1 ponors in Ljubomirsko Polje 14 km away. Although it is locally regarded as a vrelo, vruljakensis it is actually an estavelle. This was once used as a public water supply. Estavela ,,Pe¢ine” (Mostad¢i) (BiH19_12) 42.71244, Ancylus recurvus 100/0 This is a major estavelle-type outlet for the karst conduit-aquifer originating at the 18.30497 Galba truncatula 0/100 ponors in Ljubomirsko Polje. It was hydrologically inactive when sampled. Igorovo Jezero (lake) (Gorica); small lake in a collapsed cave passage with cave 42.71111, Ancylus sp. 0/9.1 springs and containing many ponors; muddy bottom (BiH19_19) The water 18.38495 Galba truncatula 0/36.4 originates from ponors in both Ljubomirsko Polje and Cibrijansko Polje. The Radix Labiate 0/9.1 ponors in and around the lake feed water underground downstream to Vrelo Sadleriana fluminensis 0/45.4

“Vruljak 2” (Gorica).

Aquatic molluscs of the Trebignjica River Basin 67

Site names, characteristics and codes Coordinates Taxa confirmed % of taxa in site (surface/ pump) Vrelo ,,Vruljak 2” (Gorica), Trebinjsko Polje; this location was sampled at the 42.71062, Kerkia briani 15.9/0 resurgence spring outlet before which 2 cave rivers have joined inside: Rijeka 18.37618 | Plagigeyeria tribunicae 2.3/0 “PeStercica’ and Rijeka “Venator” (BiH19_02) Radinianinla gurta 0/96.5 ‘This is a permanently hydrologically active outlet from a locally very complex cave Sadleriana fluminensis 0/3.5 system containing a very rich biodiversity. Tbunbicn 81.8/0 vruljakensis ‘The intermittently active cave spring, Vrelo ,,Vraziji Mlin” (D. Granéarevo); 42.70847, Radomaniola cf. 0/100 Trebignjica Canyon (BiH19_04) 18.44801 bosniaca ‘This is fed by ponors in Jasen Polje. The location is set in dolomitic limestone. “Slomljen pecina” (Mokri Dolovi); (BiH19_22) 42.70844, = - Since being sampled, this location has now been buried and made inaccessible by 18.35419 urban development. Confluence of SuSica River and Jazina River (Jazina) (BiH19_16) 42.70429, Iglica cf. absoloni 16.7/0 ‘This was sampled under low-flow conditions. The source of the water is a giant 18.50491 Litthabitella chilodia 83.3/0 estavelle situated in karstified dolomite with dolomitic limestone. Radix labiata 0/72.2 Valvata montenegrina 0/27.8 Vrelo ,,LuSac” (Guéina); at the surface outlet (BiH19_10) 42.70111, Litthabitella chilodia 14.6/0 A permanently hydrologically active outlet from a complex karst conduit-aquifer, 18.3575 | Montenegrospeum bogici| 22.0/0 whose principal source is unproven. This was once a public water supply. Pisidium cf. personatum 4.9/0 Paladilhiopsis arion 58.5/0 Travunijana 0/100 vruljakensis Estavela ,,Mali Sumet” (Bugovina), Mokro Polje: in the entrance shaft (BIH19_01)} 42.65665, | Emmericia ventricosa 0/100 ‘The entrance comprises a neo-circular stone wall leading down into the interior by 18.34458 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. River Konavoska Ljuta (Ljuta), Croatia; samples from the surface (Stones, plants) 42.53408, | Pisidium cf. personatum 15.6/0 (BiH19_18) 18.37647 This karst river originates from Vrelo “Konavoska Ljuta’ a few metres upstream from Radomaniola curta 84.4/100 the sampling location. However, the water itself originates from a ponor 10 km away in Zubacko 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.

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).

68 Andrzej Falniowski et al. / ZooKeys 1047: 61-89 (2021)

Table 2. Taxa used for phylogenetic analyses with their GenBank accession numbers and references.

Species Agrafia wiktori Szarowska & Falniowski, 2011 Algoniella finalina Giusti & Bodon, 1984 Anagastina zetavalis (Radoman, 1973) Ancylus sp. B Ancylus sp. C4 Ancylus sp. — clade 3 Ancylus sp. — clade 4

Avenionia brevis berenguieri (Bourguignat, 1882)

Belgrandia thermalis (Linnaeus, 1767) Belgrandiella kuesteri (Boeters, 1970) Belgrandiella kusceri (A. J. Wagner, 1914) Bithynia tentaculata (Linnaeus, 1758)

Bracenica gloeri Grego, Fehér & Eréss, 2020 Bythinella cretensis Schiitt, 1980

Bythiospeum acicula (Hartmann, 1821) Daphniola louisi Falniowski & Szarowska, 2000 Dalmatinella fluviatilis Radoman, 1973 Dalmatinella simonae Beran & Rysiewska, 2021 Ecrobia maritima (Milaschewitsch, 1916) Emmericia expansilabris Bourguignat, 1880 Fissuria boui Boeters, 1981

Graecoarganiella parnassiana Falniowski & Szarowska, 2011

Graecoarganiella sp.

Graziana alpestris (Frauenfeld, 1863) Grossuana hohenackeri (Kiister, 1853) Hauffenia michleri (Kuséer, 1932)

Heleobia maltzani (Westerlund, 1886) Horatia klecakiana Bourguignat, 1887 Iglica gracilis (Clessin, 1882)

Islamia zermanica (Radoman, 1973) Littorina littorea (Linnaeus, 1758) Lithoglyphus prasinus (Kiister, 1852) Marstoniopsis insubrica (Kiister, 1853) Moitessieria cf. puteana Coutagne, 1883 Montenegrospeum bogici (Pesié & Gléer, 2012) Montenegrospeum sketi Grego & Gléer, 2018 Paladilhiopsis grobbeni Kuscer, 1928 Pontobelgrandiella sp. Radoman, 1978 Pseudamnicola pieperi (Schiitt, 1980) Pseudorientalia sp.

Radomaniola curta (Kiister, 1853) Radomaniola curta curta (Kiister, 1853)

Radomaniola sp.

Sadleriana fluminensis (Kuster, 1853) Sarajana apfelbecki (Brancsik, 1888) Sarajana cf. apfelbecki

Tanousia zrmanjae (Brusina, 1866)

Gastropoda Neritopsina: Neritidae

COI/H3 GB numbers JF906762/MG543158 AF367650/- EF070616/- DQ301830 DQ301838/- KY012232 KY012163/- AY350516 AY350519/- AY350520 AY350521/- AF367638/- AF367648/- MG551325/- -/MG551366 AF367643/- MT396209/- KT353689/- KU341350/MK609536 KM887915/- KC344541/- MT773271/- KX355835/MG551322 KC810060/- AF367654/- JN202352/- JN202353/MN03140 AF367641/- KC011749/- KT236156/KY087878 KM213723/MKG609534 KJ159128/- MH720985/MH721003 KU662362/MG551320 KF644330/KP1 13574 JX073651/- AF322408/- AF367635/MH721012 KM875510/MG880218 MG880216/- MH720991/MH721014 KU497024/MG55 1321 KT710670/- KJ920477/- KCO011814/- KC011781 KC011784 KC011787 KCO011788 KC011791 KC011792 KC011810/- KC011727 KC011745 KC011747 KCO11763 KC011764 KC011766/- KF193067/- MN031432/MN031438 MNO031431/- KU041812/-

Theodoxus callosus (Deshayes, 1833)

References

Szarowska and Falniowski 2011/Grego et al. 2017

Wilke et al. 2001 Szarowska 2006 Albrecht et al. 2006 Macher et al. 2016 Pfenninger et al. 2003 Pfenninger et al. 2003 Wilke et al. 2001 Wilke et al. 2001 Osikowski et al. 2018 Osikowski et al. 2018 Wilke et al. 2001 Hofman et al. 2020a Szarowska et al. 2016b

Richling et al. 2016/Falniowski et al. 2019

Szarowska et al. 2014a Falniowski and Szarowska 2013 Beran et al. 2021 Osikowski et al. 2016/Grego et al. 2017 Szarowska and Falniowski 2013a Wilke et al. 2001 Falniowski and Szarowska 2011

Falniowski and Szarowska 2011/Hofman et al. 2019

Wilke et al. 2001 Falniowski et al. 2012

Falniowski and Szarowska 2015 /Rysiewska et al. 2017 Szarowska et al. 2014b/ Falniowski et al. 2019

Szarowska and Falniowski 2014 Hofman et al. 2018 Beran et al. 2016/Grego et al. 2017 Layton et al. 2014/unpub. Falniowski and Szarowska 2012 Falniowski and Wilke 2001 Wilke et al. 2001/Hofman et al. 2018 Falniowski et al. 2014/Grego et al. 2018 Grego et al. 2018 Hofman et al. 2018 Rysiewska et al. 2016/Grego et al. 2017 Szarowska et al. 2016a Szarowska et al. 2014c Falniowski et al. 2012 Falniowski et al. 2012

Falniowski et al. 2012

Szarowska and Falniowski 2013b Hofman et al. 2019 Hofman et al. 2019

Beran et al. 2015

Remarks. ‘This species, described from Greece and reported from Greece and Albania, was found at some larger springs, but never in subterranean waters.

Aquatic molluscs of the Trebignjica River Basin 69

Figure 4. Distribution of the studied taxa. Localities’ numbers correspond to Table 1.

Caenogastropoda Hydrobiidae: Sadlerianinae

Sadleriana fluminensis (Kiister, 1852) Fig. 5A

GenBank no. COI: MZ027620-MZ027622 Remarks. The most widely distributed species of Sadleriana. Found at the locali- ties 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) distin- guished 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, sug- gesting a flock of distinct species. The phylogeography as well as molecularly-based

70 Andrzej Falniowski et al. / ZooKeys 1047: 61-89 (2021)

Figure 5. Shells of the studied gastropods: A Sadleriana fluminensis, locality 10 B-M Radomaniola B=-H 2. 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), repre- senting 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 KCO11784), we used a provisional assignment to this species; for the second clade we provisionally used the name R. cf. bosniaca. In general, the representa- tives 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.

Aquatic molluscs of the Trebignjica River Basin 71

2G78 - Radomaniola curta - site 23 2G92 - Radomaniola curta - site 10 2G91 - Radomaniola curta - site 10 2D46 - Radomaniola curta - site 13 2G11 - Radomaniola curta - site 13 2G07 - Radomaniola curta - site 13 2G90 - Radomaniola curta - site 10 2G79 - Radomaniola curta - site 23 2G93 - Radomaniola curta - site 10 2D18 - Radomaniola curta - site 17 97/1.0| | Radomaniola curta curta Radomaniola curta curta Radomaniola curta curta Radomaniola curta curta

95/0.99

74/0.82|°"""”3], Radomaniola curta curta Radomaniola curta curta * 89/0.92L— Radomaniola curta curta *

2G75 - Radomaniola cf. bosniaca - site 14 2D30 - Radomaniola cf. bosniaca - site 6 2C79 - Radomaniola cf. bosniaca - site 12 76/0.80 2C81 - Radomaniola cf. bosniaca - site 12 2C80 - Radomaniola cf. bosniaca - site 12 96/1.0| f 2D06 - Radomaniola cf. bosniaca - site 1 2D31 - Radomaniola cf. bosniaca - site 1 2D24 - Radomaniola cf. bosniaca - site 12 94/0.98 |- 2D36 - Radomaniola cf. bosniaca - site 1 2G08 - Radomaniola cf. bosniaca - site 13 2D01 - Radomaniola cf. bosniaca - site 18 2D22 - Radomaniola cf. bosniaca - site 12 2D07 - Radomaniola cf. bosniaca - site 1 2D12 - Radomaniola cf. bosniaca - site 1 2D11 - Radomaniola cf. bosniaca - site 1 2D08 - Radomaniola cf. bosniaca - site 1 2D14 - Radomaniola cf. bosniaca - site 1 2D33 - Radomaniola cf. bosniaca - site 1 2D15 - Radomaniola cf. bosniaca - site 1 Radomaniola sp. Radomaniola sp. Radomaniola sp. 91/0.95 Radomaniola sp. Radomaniola sp. 100/1.0'"— Radomaniola sp. Anagastina zetavalis Dalmatinella fluviatilis

79/0.84

76/0.80

0.050 Figure 6. Maximum likelihood (ML) phylogram of the studied Radomaniola, based on the partial cy-

tochrome oxidase subunit I (COI) sequences, bootstrap supports given if >60%, together with Bayesian probabilities; topotypes of R. curta curta marked with asterisks.

TD Andrzej Falniowski et al. / ZooKeys 1047: 61-89 (2021)

Radomaniola curta (Kiister, 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 Policki 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; Hof- man 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 (Pesi¢é & Gléer, 2012) Fig. 7D-K

GenBank no. COI: MZ266648—MZ266650

Remarks. Pesi¢ and Gléer (2012) described a new species of Bythiospeum Bour- guignat, 1882: B. bogici Pesi¢ & Gloéer, 2012 from underground waters of Vrelo “Taban”, in central Montenegro. Their description was based on empty shells. Later they (Pesi¢ and Gléer 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 Pesié & Gloer, 2013. Later, Falniowski et al. (2014) demonstrated with molecular data that Montenegrospeum belongs to the Hy- drobiidae, not Moitessieriidae, despite striking similarity of the shell between this snail and e.g., /glica Wagner, 1927. Numerous live specimens of this species were pumped from interstitial habitats at the localities 1, 7 and 21 (Fig. 4).

Aquatic molluscs of the Trebisnjica River Basin 73

Figure 7. Shells of the studied gastropods: A-C Kerkia briani D-K Montenegrospeum bogici (localities: D-F — 1, G,H —7, | - 13, J— 14, K-21) L-O Litthabitella chilodia (localities: L, M — 17, N—O — 8).

Scale bars: 1 mm.

74 Andrzej Falniowski et al. / ZooKeys 1047: 61-89 (2021)

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 & Gléer, 2019 Bier)

GenBank no. COI: MW879256-MW879272; H3: MW865737-MW 865748

Remarks. Grego and Gléer (2019) described a new monotypic genus Travunijana from Vrelo “Goricki Studenac” (Gorica), a spring at the right bank of the TrebiSnjica River, this being its type locality. They found it also in two other springs: Vrelo Vrul- jak 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 pe- nis — which was based on artefactual appearance, caused by fixation: a nonglandular outgrowth on the left side, located distally (Grego and Gléer 2019). The penis pho- tographed by them presents a bulbous, drastically contracted distal section, making copulation impossible.

Our molecular data (Fig. 8) confirmed the distinctiveness of the genus Travuni- jana Grego & Gléer, 2019. The phylograms based on H3, as well as on both concat- enated 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 & Gléer, 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 Gloer (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, of Radoman 1973) and distal (rs, of Radoman 1973) one. The penis

Aquatic molluscs of the Trebi8njica River Basin 75

2426 - Travunijana vruljakensis - BiH - site 17 2J28 - Travunijana vruljakensis 2J24 - Travunijana vruljakensis 2423 - Travunijana vruljakensis 2J21 - Travunijana vruljakensis 2F73 - Travunijana vruljakensis - BiH - site 17 2F72 - Travunijana vruljakensis - BiH - site 17 2F66 - Travunijana vruljakensis - BiH - site 13 2F60 - Travunijana vruljakensis - BiH - site 13 2034 - Travunijana vruljakensis - BiH - site 12 2C71 - Travunijana vruljakensis - BiH - site 17 2618 - Travunijana vruljakensis - BiH - site 13 2G76 - Travunijana vruljakensis - BiH - site 14 2G77 - Travunijana vruljakensis - BiH - site 14 2CT4 - Travunijana vruljakensis - BiH - site 12 — sonal 2CT3 - Travunijana vruljakensis - BiH - site 17 2C75 - Travunijana vruljakensis - BiH - site 17 soft 2F61 - /glicopsis - site 13 | 2F68 - Iglicopsis - site 13

2F69 - Iglicopsis - site 13

Daimatinella simonae 97/121 Dalmatinella fluviatilis Belgrandia thermalis Montenegrospeum sketi

99/10" Montenegrospeum bogici Tanousia zrmanjae

Sarajana cf. apfelbecki Sarajana apfelbecki

[= Graecoarganiella parnassiana 85/0.90 Graecoarganiella sp. Anagastina zetavalis Radomaniola curta Grossuana hohenackeri Daphniola louisi Pseudorientalia sp. Horatia klecakiana Sadleriana fluminensis '—— Bracenica gloeri Agratia wiktori Hauffenia michleri Belgrandiella kuesteri Graziana alpestris Pontobelgrandiella sp. Fissuria boui islamia zermanica Alzoniella finalina Avenionia brevis berenguieri Ecrobia maritima Pseudamnicola pieperi Heleobia malzani Bythiospeum acicula Moitessieria cf. puteana Iglica gracilis Paladilhiopsis grobbeni

Lithoglyphus prasinus Bithynia tentaculata —————— Bythinella cretensis Emmericia expansilabris Marstoniopsis insubrica

Littorina littorea

72/078

aos

Col

2C75 - Travunijana vruljakensis - BiH - site 17 2F72 - Travunijana vruljakensis - BiH - site 17 2C73 - Travunijana vruljakensis - BiH - site 17 2C71 - Travunijana vruljakensis - BiH - site 17 2G77 - Travunijana vruljakensis - BiH - site 14 100/1.0| 2G76 - Travunijana vruljakensis - BiH - site 14 2618 - Travunijana vruljakensis - BiH - site 13 2F65 - Travunijana vruljakensis - BiH - site 13 2F60 - Travunijana vruljakensis - BiH - site 13 2C74 - Travunijana vruljakensis - BiH - site 12 2034 - Travunijana vruljakensis - BiH - site 12 2F73 - Travunijana vruljakensis - BiH - site 17 Graecoarganiella sp.

Sarajana apfelbecki

Say 2F69 - Iglicopsis - site 13

0.09 2F68 - Iglicopsis - site 13

2F61 - Iglicopsis - site 13

Montenegrospeum bogici

Belgrandiella kuesteri

Agrafia wiktori

Hauffenia michleri

Pontobelgrandiella sp.

Islamia zermanica

Ecrobia maritima

Heleobia maltzani

Bythiospeum acicula

Moitessieria sp. puteana

Iglica cf. gracilis

Paladilhiopsis grobbeni

62/0.65

610.64

Lifforina littorea

bos

H3

2F65 - Travunijana vruljakensis - BiH - site 13 2F72 - Travunijana vruljakensis - BiH - site 17 2F60 - Travunijana vruljakensis - BiH - site 13 2034 - Travunijana vruljakensis - Bi i 2F73 - Travunijana vruljakensis - BiH - si’

2C71 - Travunijana vruljakensis - BiH - site 17

92/0.95

60/0.67

2G76 - Travunijana vruljakensis - BiH - site 14 74/0.76'2G77 - Travunijana vruljakensis - BiH - site 14 Graecoarganiella parnassiana

Sarajana apfelbecki

2F69 - iglicopsis - site 13

2F68 - iglicopsis - site 13

2F61 - /glicopsis - site 13

Montenegrospeum bogici

Belgrandiella kuesteri

Agrafia wiktori Hauffenia michleri

Islamia zermanica Pontobelgrandielia sp. Ecrobia maritima Heleobia malzani Bythiospeum acicula Moitessieria cf. puteana

Iglica cf. gracilis Paladilhiopsis grobbeni

Littorina littorea

0050

COl + H3

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 locali- ties 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-D5632F9E5F5 1

Type species. /¢licopsis 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 bi- lobed outgrowth on the left side and flat outgrowth at the right side, unpigmented renal oviduct, bursa copulatrix and two small receptacula seminis.

Remarks. Jg/icopsis shell resembles that of Montenegrospeum, but is more oval, with broader spire and broader flat apex, sometimes showing scalarity at the body

76 Andrzej Falniowski et al. / ZooKeys 1047: 61-89 (2021)

Figure 9. Shells: A-L Travunijana vruljakensis M=-P Ielicopsis butoti M holotype N 2F61 O 2F68 P 2FG69 (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.

Aquatic molluscs of the Trebignjica River Basin es

Figure 10. Female reproductive organs of Travunijana vruljakensis (bc — bursa copulatrix, cbc — duct of bursa, ga — albuminoid gland, gn — nidamental gland, gp — gonoporus, ov — oviduct, ovl — loop of (renal)

oviduct, rs, — distal seminal receptacle, rs, — proximal seminal receptacle). Scale bar: 0.25 mm.

Iglicopsis butoti Falniowski & Hofman, sp. nov. http://zoobank.org/C1A9DOBO0-4B 10-4977-B69B-7C4C42BB 19D3 Fig. 9M—P

GenBank no. COI: MW879273-MW879275; H3: MW865749-MW 86575 1

Type materials. Holotype. Ethanol-fixed specimen (Fig. 9M), Vrelo ,,Policki Stu- denac” (Crkvina); a cave spring in the left bank of and adjacent to the TrebiSnjica 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 col- lection of the Department of Malacology of Jagiellonian University, voucher number ZMUJ-M.2651.

Paratypes. Three paratypes destroyed to extract DNA, one specimen ethanol- fixed, 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.

78 Andrzej Falniowski et al. / ZooKeys 1047: 61-89 (2021)

Table 3. Shell measurements (in mm) of holotype and sequenced and illustrated specimens of Iglicopsis butoti sp. nov. For explanation of the symbols a-f, 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 om 90 89 90 90 B 20 18 20 18

Figure I 1. Penis of Travunijana vruljakensis. Scale bars: 0.5 mm.

Description. Shell (Fig. 9M—P) up to 1.49 mm high and 0.55 mm broad, ovate- conic, 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 vari- ability 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. Ctenid- ium 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) ie (5)4—1-4(5) 1-1 1-1

Aquatic molluscs of the Trebignjica River Basin

Figure 12. Radula of Jglicopsis butoti, scale bars: 10 um.

80 Andrzej Falniowski et al. / ZooKeys 1047: 61-89 (2021)

Figure 13. A Penis of /glicopsis butoti, scale bar: 0.1 mm B shell measurements: a — shell height, b — body

whorl breadth, c— aperture height, d— spire height, e— aperture breadth, « — apex angle, 8 — angle between

body whorl suture and horizontal surface.

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 invari- able 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 /g/ica Wagner, 1927. The specific epithet butoti refers to the mem- ory of Dr Louis J. M. Butot, a Dutch malacologist devoted mostly to the Greek mala- cofauna, good friend and the mentor of AF.

Distribution and habitat. Known from the type locality only.

Molecular relationships. despite its shell morphology, Jelicopsis clearly belongs to the Hydrobiidae Stimpson, 1865, Sadlerianinae Szarowska, 2006, and not to the

Aquatic molluscs of the Trebisnjica River Basin 81

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 Schitt, 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 local- ity 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 Pavlovi¢, 1913. Our specimens from Gu¢ina in Trebinje molecu- larly formed the sister clade with Plagigeyeria montenegrina Bole, 1961 from Obodska Pecina 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 Schiitt, 1963, but the number of whorls of our speci- mens is much higher than presented by Schiitt (1972).

82 Andrzej Falniowski et al. / ZooKeys 1047: 61-89 (2021)

Heterobranchia Heterostropha: Valvatidae

Valvata montenegrina Gléer & Pesic, 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 (Rossmissler, 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., Gléer 2019), preferably close to ground waters, but not found in subter- ranean habitats.

Galba truncatula (O. F. Miiller, 1774) Fig. 14H, I

GenBank no. COI: MZ027628-MZ027629

Remarks. Common Palaearctic gastropod, inhabiting nearly all of Europe. This am- phibious and calcifilous (e.g., Gléer 2019) species inhabits small water bodies, rich in veg- etation, 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 tun- nel 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., Ro- szkowski 1914; Falniowski 1980, 1981), as being wider than in any other gastropod group.

Ancylidae Ancylus recurvus Martens, 1783

Fig. 14], 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

Aquatic molluscs of the Trebisnjica River Basin 83

Figure 14. Shells of the studied gastropods: A-C Emmericia ventricosa (localities: A — 1, B—5, C — 12) D, E Paladilhiopsis arion (locality 21) F Valvata montenegrina (locality 20) G Radix labiata (locality 16) H, | Galba truncatula (localities: H — 15, | — 16) J, K Ancylus recurvus (localities: J-— 15, K — 13) L, M Ancylus sp. C4 (localities: L— 9, M — 16) N Succinea cf. putris (locality 10). Scale bars: 1 mm.

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 molecu- larly different from A. fluviatilis by 9%.

84 Andrzej Falniowski et al. / ZooKeys 1047: 61-89 (2021)

2G17 - Ancylus recurvus - site 13 2G72 - Ancylus recurvus - site 15 2F66 - Ancylus recurvus - site 13 Ancylus sp. B

Ancylus sp. B

Ancylus sp. - clade 3 Ancylus sp. - clade 3

100/1.0, 2G44 - Ancylus sp. - site 8

2G80 - Ancylus sp. - site 9

Ancylus sp. C4 Ancylus sp. C4 Ancylus sp. - clade 4 100/1.0 " Ancylus sp. - clade 4

91/0.95

93/0.98

99/1.0

99/1.0

Ancylus fluviatilis

0.020

Figure 15. Molecular relationships of the studied Ancylus based on COI; our sequences in red and orange, the other from GenBank; bootstrap supports given if over 60%, their values together with Bayesian probabilities.

Ancylus sp. Fig. 14L, M

GenBank no. COI: MW879254-MW879255

Remarks. Considering the shell morphology, it should be determined as A. fluvia- tilis O. F. Miller, 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

Aquatic molluscs of the Trebignjica River Basin 85

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.

References

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