Zospeum sp. 2

Jochum, Adrienne, Michalik, Peter, Inäbnit, Thomas, Kneubühler, Jeannette, Slapnik, Rajko, Vrabec, Marko, Schilthuizen, Menno & Ruthensteiner, Bernhard, 2024, 3 D X-ray microscopy (Micro-CT) and SEM reveal Zospeum troglobalcanicum Absolon, 1916 and allied species from the Western Balkans (Ellobioidea: Carychiidae), European Journal of Taxonomy 926, pp. 1-62 : 51-53

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.

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.

Figures

Fig. 20.3D visualizations of Micro-CT data ofZospeum 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.

Fig. 2. 3D visualizations of Micro-CT data of the lectotype of Zospeum troglobalcanicum Absolon, 1916 (NHMW Moll.Coll.Edlauer 32.749) with shell parts and terms indicated. A. Measurements include shell height (sh), shell width (sw), aperture height (ah), aperture width (aw), height of last whorl (hlw) and spire angle (SA). B. Internal view showing columella (co), lamella (la), peristome (pe) and penultimate whorl (pw). C. Apical view showing protoconch (pr), teleoconch (te) and peristome (pe). D. Ventral view showing peristome configuration (pe) parietal shield (ps) and umbilicus (um).

Fig. 3. Light microscopic images of shells from the caves Benetina pećina (SE Bosnia and Herzegovina) and Cetinska pećina (Montenegro) with specimen labels. A. Zospeum troglobalcanicum Absolon, 1916, lectotype (NHMW Moll.Coll.Edlauer 32.749) from Benetina pećina. B–C. Zospeum tortuosum Jochum & Ruthensteiner sp. nov., paratypes (MCSMNH-PMSL-Moll.-FVelkovrh 34099[spm2-3]) from Cetinska pećina. D. Zospeum tortuosum Jochum & Ruthensteiner sp. nov., holotype (MCSMNH- PMSL-Moll.-FVelkovrh 34099[spm1]) from Cetinska pećina. E. Deformed shell (MCSMNH-PMSL- Moll.-FVelkovrh 34099[spm4]) from Cetinska pećina.

Fig. 4. 3D visualizations using Micro-CT data of shells from the caves Benetina pećina (SE Bosnia and Herzegovina) and Cetinska pećina (Montenegro). A–F. Zospeum troglobalcanicum Absolon, 1916, lectotype (NHMW Moll.Coll.Edlauer 32.749). A. Aperture view. B. Aperture facing right view. C. Apical view. D. Dorsal view. E. Aperture facing left view. F. Ventral view showing umbilical depression. G–L. Zospeum tortuosum Jochum & Ruthensteiner sp. nov., holotype (MCSMNH-PMSL- Moll.-FVelkovrh 34099[spm1]). G. Aperture view. H. Aperture facing right view. I. Apical view. J. Dorsal view. K. Aperture facing left view. L. Ventral view showing callused peristome positioned mostly on top of umbilicus.

Fig. 5. 3D visualizations of Micro-CT data of the deformed shell, (MCSMNH-PMSL-Moll.-FVelkovrh 34099[spm4]) from Cetinska pećina. A. Aperture view. B. Aperture facing right view. C. Apical view. D. Dorsal view. E. Aperture facing left view. F. Ventral view with umbilicus covered by last quarter of shell whorl.

Fig. 6. Zospeum intermedium Jochum & Ruthensteiner sp. nov. (RMNH.MOL.234132) from Gittenberger (1975). A. Light microscopic images of apertural and dorsal views. B. Sample labels. C–H. 3D visualizations of X-ray Micro-CT data. C. Aperture view. D. Aperture facing right view. E. Apical view. F. Dorsal view. G. Aperture facing right view. H. Umbilical view showing slight umbilical depression and lamella projecting from the columellar side.

Fig. 7. Lightmicroscopic images of shells from the caves Lipska pećina and Duboki do (Montenegro) with specimen labels. A–C. Zospeum troglobalcanicum Absolon, 1916 (MCSMNH-PMSL-Moll.-FVelkovrh 29603). D–F. Zospeum dubokidoense Jochum & Ruthensteiner sp. nov. D. Holotype (MCSMNH- PMSL-Moll.-FVelkovrh 30360[spm1]). E–F. Paratypes, Duboki do, Montenegro (MCSMNH-PMSL- Moll.-FVelkovrh 30360[spm2-3]).

Fig. 8. 3D visualizations of Micro-CT data of shells from the caves Lipska pećina and Duboki do (Montenegro). A–F. Zospeum troglobalcanicum Absolon, 1916 (MCSMNH-PMSL-Moll.-FVelkovrh 29603) (Fig. 7A). A. Aperture view. B. Aperture facing right view. C. Apical view. D. Dorsal view. E. Aperture facing left view. F. Ventral view showing reduced umbilical depression. G–L. Zospeum dubokidoense Jochum & Ruthensteiner sp. nov., holotype (MCSMNH-PMSL-Moll.-FVelkovrh 30360[spm1]). G. Aperture view. H. Aperture facing right view. I. Apical view. J. Dorsal view. K. Aperture facing left view. L. Ventral view showing no umbilical depression.

Fig. 9. Zospeum amplioscutum Jochum & Ruthensteiner sp. nov., (NHMW Mol.Coll.Edlauer 19.069) with sample labels. A. Light microscopic images of paratype (NHMW Mol.Coll.Edlauer 19.069), apertural and dorsal views.B. Light microscopic images of holotype (NHMW Mol.Coll.Edlauer 19.069), aperture and dorsal views. C–H. 3D visualizations of X-ray Micro-CT data of holotype (NHMW Mol. Coll.Edlauer 19.069). C. Aperture view. D. Aperture facing right view. E. Apical view. F. Dorsal view. G. Aperture facing left view. H. Ventral view showing coiling projecting high up on shell and peristome oblique to umbilicus.

Fig. 10. Zospeum tumidum Jochum, Schilthuizen & Ruthensteiner sp. nov., holotype (NMBE 572615). A. Light microscopic images, showing aperture and dorsal views and TxEx Collection label. B. 3D visualizations of Micro-CT data, apical view. C. Ventral view showing deep umbilical depression. D. Aperture view.E. Aperture facing right view showing tapered columella and lamellar bulge. F. Dorsal view. G. Aperture facing left view.

Fig. 11. Zospeum njunjicae Jochum, Schilthuizen & Ruthensteiner sp. nov. A–B. Light microscopic images of paratypes (NMBE 572616) showing aperture and dorsal views. C. Light microscopic images of holotype (NMBE 572617) showing aperture and dorsal views. D–I. 3D visualizations of X-ray micro- CT data of holotype (NMBE 572617). D. Aperture view. E. Aperture facing right view showing well defined lamella. F. Apical view. G. Dorsal view. H. Aperture facing left view. I. Ventral view.

Fig. 12. Zospeum neuberti Jochum & Ruthensteiner sp. nov. (NHMW Mol.Coll.Edlauer 32006). A–B. Light microscopic images of paratypes showing aperture and dorsal views and collection labels. C. Light microscopic images of holotype showing aperture and dorsal views. D–I. 3D visualizations of Micro-CT data of holotype. D. Aperture view. E. Aperture facing right view showing smooth columella. F. Apical view.G. Dorsal view showing smooth columella. H. Aperture facing left view. I. Ventral view.

Fig. 13. Light microscopic images of Zospeum constrictum Jochum & Ruthensteiner sp. nov. (NHMW Mol.Coll.Edlauer 16.693) and collection labels.A. Holotype, aperture, and dorsal views.B–D. Paratypes, aperture and dorsal views.

Fig. 14. 3D visualizations of Micro-CT data of Zospeum constrictum Jochum & Ruthensteiner sp. nov. (NHMW Mol.Coll.Edlauer 16.693). A–F. Holotype. A. Aperture and dorsal views. B. Aperture facing right view. C. Apical view showing upper right side of final whorl tucked underneath spire. D. Dorsal view. E. Aperture facing left view. F. Ventral view. G–L. Paratype (Fig. 13B). G. Aperture view. H. Aperture facing right view. I. Apical view showing upper right side of final whorl tucked underneath spire. J. Dorsal view. K. Aperture facing left view. L. Ventral view.

Fig. 15. Zospeum biokovoense Jochum & Ruthensteiner sp. nov. (NHMW Mol.Coll.Edlauer 16.390). A–B. Light microscopic images showing aperture and dorsal views and collection labels. A. Juvenile shell. B. Holotype. C–H. 3D visualizations of Micro-CT data of holotype. C. Aperture view showing gnarled deformation of the columella. D. Aperture facing right view showing fat columella and lamella. E. Apical view. F. Dorsal view showing lopsided spire and incomplete lamella on top of fat columella. G. Aperture facing left view. H. Ventral view showing puckered umbilical depression.

Fig. 16. Light microscopic images of full-bodied Zospeum Bourguignat, 1856 from Njeguši, St John’s cave. A–B. Zospeum kolbae Jochum, Inäbnit, Kneubühler & Ruthensteiner sp. nov. (NMBE 571122– 571123), individuals assessed by DNA sequencing with sigmoid intestine showing through shells (shells were destroyed post imaging for tissue extraction). A. Holotype (NMBE 571122), shell of aliquot, aperture, and aperture facing left view. B. Paratype (NMBE 571123), shell of aliquot, aperture, and aperture facing left view. C. Undescribed Zospeum sp. 1 (NMBE 577052) showing all perspectives. D–E. Undescribed Zospeum sp. 1 (NMBE 577053/2) showing all perspectives.

Fig. 17. Z. njegusiense Jochum & Ruthensteiner sp. nov., Scanning Electron Microscopy images. A. Holotype (NMBE 572639), aperture view showing subfossil shell with superficial erosion of shell layer. B. Paratype (NMBE 578378), aperture view showing close up of lamellar band on columella (Fig. C). C. Paratype (NMBE 578378), aperture view showing damaged shell and columella. D. Close up view of holotype (NMBE 572639), showing body whorl with low, irregular growth lines. E. Paratype (NMBE 578378), protoconch.

Fig. 18. 3D visualizations of Micro-CT data of Zospeum simplex Inäbnit, Jochum & Neubert, 2021, (NMHW-MO-113642 ex RSC 3760) from Dobravljevac jama, Gornji Brišnik, Bosnia and Herzegovina. A–F. Specimen 1 (spm1). A. Aperture view. B. Aperture facing right view. C. Apical view. D. Dorsal view. E. Aperture facing left view. F. Ventral view. G–L. Specimen 2 (spm2). Same perspectives as Specimen 1.

Fig. 19. Zospeum simplex Inäbnit, Jochum & Neubert, 2021, (SMF 349425) Scanning Electron Microscopy images. A. Protoconch and upper teleoconch showing microstructure of superficial pitting. B. Close up view of pitting microstructure. C. Last whorl with axial ribbing extending beyond peristome lip. D. Close up view of second whorl showing rows of interrupted dashes of radial pitting. E. Growth lines and radial banding on teleoconch. F. Close up view of growth lines and shell microstructure.

Fig. 21. Bayesian tree of the genus Zospeum Bourguignat, 1856. Node support values of both the Bayesian Inference (left) and the Maximum Likelihood analysis (right) are given. Branches are coloured to denote the informal species groups within the eastern radiation of Zospeum following Inäbnit et al. (2019). Emboldened specimen names indicate samples sequenced for this study.