3 D X-ray microscopy (Micro-CT) and SEM reveal Zospeum troglobalcanicum Absolon, 1916 and allied species from the Western Balkans (Ellobioidea: Carychiidae) Author Jochum, Adrienne nne.jochum@senckenberg.de Author Michalik, Peter Author Inäbnit, Thomas 95F0148B-E147-4EB9-903A-6EEA7F55EB47 Institute for Biochemistry & Biology, University of Potsdam, Karl-Liebknecht-Strasse 24 - 25, House 26, D- 14476 Potsdam, Germany. inaebnit.thomas@gmail.com Author Kneubühler, Jeannette A42A773F-9C7C-4681-B861-B354A330DC4F Naturhistorisches Museum Bern, Bernastrasse 15, CH- 3005 Bern, Switzerland. & Institute of Ecology and Evolution, University of Bern, Baltzerstrasse 6, CH- 3012 Bern, Switzerland. jeannette.kneubuehler@nmbe.ch Author Slapnik, Rajko 09E86269-966B-49E6-AB92-31B66968DC0D ZOSPEUM, Molluscs, Cave & Karst Biological Consulting, Drnovškova pot 2, Mekinje, SI 1240 Kamnik, Slovenia. rajko.slapnik@gmail.com Author Vrabec, Marko 00C5A26B-6E4A-41D9-BC9B-F36A2799E1AF Department of Geology, Faculty of Natural Sciences and Engineering, Aškerčeva 12, University of Ljubljana, 1000 Ljubljana, Slovenia. marko.vrabec@geo.ntf.uni-lj.si Author Schilthuizen, Menno 683D0AB7-CDD8-4FAA-94B6-436F3BFB8873 Naturalis Biodiversity Center and Leiden University, Vondellaan 55, NL- 2332 AA Leiden, The Netherlands. menno.schilthuizen@naturalis.nl Author Ruthensteiner, Bernhard 9A8A56B0-19FA-42AB-A85E-4FCA98BF4A6F SNSB - Zoologische Staatssammlung München (ZSM), 81247 München, Germany. ruthensteiner@snsb.de text European Journal of Taxonomy 2024 2024-03-20 926 1 62 https://europeanjournaloftaxonomy.eu/index.php/ejt/article/download/2469/10973 journal article 10.5852/ejt.2024.926.2469 2118-9773 10847806 A00D7669-2151-46AE-A066-9AF4D0F2BEEE Zospeum sp. 2 Fig. 20 Zospeum troglobalcanicum – Inäbnit et al. 2019: 19 , fig. 7u. Diagnosis (from Inäbnit et al. 2019 ) Shell ca 1.3 mm , transparent, with a globose conical form, peristome with well-defined parietal shield, totally smooth columella, lamellae missing completely. Material examined BOSNIA AND HERZEGOVINA • Trebinje , Taleža , “Trebinjska šuma” [Trebinje Forest], Taleža pećina ; 42.7097° N , 18.2430° E ; 9 Aug. 2010 ; M. Lukić leg.; NMBE 553414/1 (ex RSC 1981) . Description (using Micro-CT image data) MEASUREMENTS (Micro-CT). sh: 1.30 mm ; sw: 1.00 mm; ah: 0.58 mm ; aw: 0.60 mm ; hlw: 0.83 mm ; SA: 71.60 deg. Shell ca 1.3 mm , conical, with 5¼ regularly coiled, flatly convex whorls; transparent when fresh; suture uneven and shallow; height of last whorl greater than half of shell height; aperture reniform, narrow, slightly wider than high; teleoconch sculpture with some irregular blunt growth lines; suture zone of penultimate and final whorl with indentations from which growth lines extend irregularly; peristome thin and flattened at upper palatal side, with thickened callused zone starting after ca ⅓ the length of the palatal side; columellar side of peristome slightly oblique and callused, palatal and basal palatal edge narrowly reflected; parietal shield well-defined, thickly callused, long and straight with notch at upper parietal and palatal junction; upper rim of peristome recedes ca 1/5 the width of the penultimate whorl (aperture facing left); columella centrally aligned and totally smooth, lower part of internal penultimate whorl shows a low ridge-like fold independent of and above the formation of the columella but not leading onto it; ventral side with a shallow umbilical depression with some puckering of blunt ridges leading into it behind the columellar side of the peristome; columellar side of peristome positioned directly above umbilical depression; alignment of last 1 / 5 whorl somewhat compact. Distribution Taleža pećina is located in the Trebinjska šuma region, a 23 km long karst rocky plateau of eastern Bosnia and Herzegovina . Remarks Inäbnit et al. (2019) compared this shell to the Western Balkan congeners in their molecular study of the radiation of Dinaride Zospeum , concluding that it mostly shared an overall resemblance to the photograph of Z. troglobalcanicum supplied by Absolon (1916b) . This conclusion was based on the globose conical form of the shell, the peristome with a well-defined parietal shield and that lamellae were lacking completely. Though Inäbnit et al. (2019) considered this morph Z. troglobalcanicum , it differs from the lectotype by the smooth columella and the configuration of the umbilical zone, including the shallow umbilical depression and the relatively tight alignment of the columellar side of the peristome in relation to it. It is also much smaller than Z . troglobalcanicum , constituting one of the smallest morphs in this study, except for Z. njunjicae Jochum, Schilthuizen & Ruthensteiner sp. nov. , which on the average, bears the smallest shell. This shell is very similar to Z. neuberti Jochum & Ruthensteiner sp. nov. in that the columella is smooth, albeit somewhat attenuate, with the umbilical zone bearing similar morphology. The spire is wider, the aperture width is broader and there are fewer low ribs behind the peristome compared to the three individuals of Z. neuberti from the type locality, Grabovica cave. Due to the lack of comparative material from Taleža pećina, no conclusive conchological determination can be made based on this one shell. Fig. 20. 3D visualizations of Micro-CT data of Zospeum sp. 2 , Taleža pećina, bei Trebinje, BiH, presented as Z. troglobalcanicum in Inäbnit et al. 2021 (NMBE 553414/1 (ex RSC 1981). A . Aperture view showing low, ridge-like fold on inner penultimate whorl. B . Aperture facing right view showing smooth columella. C . Apical view. D . Dorsal view. E . Aperture facing right view. F . Ventral view showing peristome edge directly parallel to umbilical depression. Molecular analysis Zospeum troglobalcanicum is revealed based on a single syntype shell discovered in the NHMW collection and morphologically assessed along with shells deriving from 15 populations of southern Balkan Zospeum from both museum collections and recent sampling efforts ( Figs 2–19 ). Two recently collected individuals from one population from Njeguši, Montenegro were additionally assessed by DNA sequencing in accordance with the most recent integrative study by Inäbnit et al . (2021) . The phylogeny is updated in this work ( Fig. 21 ). Tree topologies in both the ML and the BI tree were identical to each other and largely congruent to the ones published in Inäbnit et al . (2021) . The two new specimens from Njeguši, Montenegro ( Z. kolbae Jochum, Inäbnit, Kneubühler & Ruthensteiner sp. nov. NMBE 571122–571123), were recovered as the sister group of Z. aff. troglobalcanicum from Špilja Jezero in southernmost Croatia . The ABGD analysis for the Z. pretneri -group yielded two different topologies, one with six clades (Prior Maximal Distance P = 3.59 e- 02; barcode gap distance: 0.042) and one with eight clades (Prior Maximal Distance P = 4.64 e- 03; barcode gap distance: 0.004). Both topologies regard the species from Njeguši, Montenegro , as a distinct species. The topology with six clades corresponds to the one that was ultimately used for species delimitation in Inäbnit et al. (2021) and which accepts all currently accepted species within the group as independent lineages, while the topology with eight clades subdivides Z. simplex and Z. aff. troglobalcanicum into two lineages each (with the two lineages in the latter co-occurring in the same cave). We will use the topology with six clades from now on.