Research Article |
Corresponding author: Adrienne Jochum ( adrienne.jochum@gmail.com ) Academic editor: Oana Teodora Moldovan
© 2015 Adrienne Jochum, Rajko Slapnik, Annette Klussmann-Kolb, Barna Páll-Gergely, Marian Kampschulte, Gunhild Martels, Marko Vrabec, Claudia Nesselhauf, Alexander M. Weigand.
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.
Citation:
Jochum A, Slapnik R, Klussmann-Kolb A, Páll-Gergely B, Kampschulte M, Martels G, Vrabec M, Nesselhauf C, Weigand AM (2015) Groping through the black box of variability: An integrative taxonomic and nomenclatural re-evaluation of Zospeum isselianum Pollonera, 1887 and allied species using new imaging technology (Nano-CT, SEM), conchological, histological and molecular data (Ellobioidea, Carychiidae). Subterranean Biology 16: 123-165. https://doi.org/10.3897/subtbiol.16.5758
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The minute troglobitic species, Zospeum isselianum Pollonera, 1887 (Eupulmonata: Ellobioidea, Carychiidae) is widely distributed within its Southern Alpine-Dinaric range. Its broad distribution and highly variable shell has caused this species to be historically lumped into its current taxonomic state of ambiguity. In an integrative taxonomic approach, phenotypic and genotypic data are synthesized to assess the intraspecific variability recently inferred for this taxon. We collected 16 Zospeum specimens in the Slovenian Alpine Arc encompassing the type locality for Z. isselianum. The material comprises five morphologically recognized (sub)species. The species are re-evaluated using SEM, X-ray nanotomography (nano-CT), conchological, histological and molecular data.
Four well-defined lineages are present, which can be attributed to i) Z. isselianum s.str. from its new type locality (Turjeva jama), ii) a highly morphologically variable lineage that so far cannot be attributed to a single morphospecies, iii) Z. kupitzense A. Stummer, 1984 (raised to species rank) and iv) a lineage comprising the two subspecies Z. alpestre alpestre (Freyer, 1855) and Z. alpestre bolei Slapnik, 1991 plus Z. isselianum individuals. The latter is treated as a single taxon Z. alpestre. After considering the severely degraded syntype material of Zospeum isselianum, we provide a taxonomic re-description and propose a neotype for this species. Furthermore, new diagnostic information is revealed regarding the columella of Zospeum isselianum and allied species. Detailed anatomical study reveals new structural aspects of Zospeum morphology and provides groundwork for future investigations.
Neotypification, lumped species, subterranean microgastropods, Byne’s degradation, species flock
We especially wish to dedicate this work to the Slovenian malacologist, Jože Bole in commemoration of the 20th anniversary of his death (December 25, 1995). His immense contributions to malacology, especially in Zospeum research, have provided the knowledge base upon which we rely today.
Systematic research is undergoing a major transition in the assortment of tools and terminology now available in today’s taxonomic toolbox. In addition, species delimitation, the practice of recognizing and determining species boundaries, has expanded to evaluate the current inflation of species concepts generated to infer biological information (de Queiroz 2000,
In this work, we conduct the requisite transition from molecular species delimitation approaches (
Zospeum is known to inhabit caves of Northern Spain to the Balkan Dinarides. Most species were described in the latter half of the 19th and 20th Centuries based on shell characters such as whorl number, aperture dentition, and shell size as well as shape and number of lamellae circumscribing the columella (
Six anatomical studies provide the current knowledge upon which morphological findings of this investigation are based (
Past anatomical investigations have involved intrepid and remarkable dissections of some now dubious, Zospeum designations (
Zospeum isselianum has up to now, represented a Southern Alpine and Dinaric taxon (
Material is housed in the following collections:
CBSS Croatian Biospeleological Society, Zagreb, Croatia
CSR SASA Centre for Scientific Research of the Slovenian Academy of Sciences and Arts in Ljubljana, Slovenia
MSNG Museo Civico di Storia Naturale »Giacomo Doria«, Genoa, Italy
MHNG Museum d'Histoire Naturelle de Genève, Geneva, Switzerland
MZUT Museo Regionale di Scienze Naturali Sezione di Zoologia, Torino, Italy
NHMW Naturhistorisches Museum Wien, Vienna, Austria
NMBE Naturhistorisches Museum der Burgergemeinde Bern, Bern, Switzerland
RS Rajko Slapnik malacological collection, Kamnik, Slovenia
SMF Senckenberg Forschungsinstitut und Naturmuseum, Frankfurt am Main, Germany
SMNH Slovenian Museum of Natural History Ljubljana, Slovenia
Material was collected from caves of the Julian Alps (Turjeva jama) and the Kamnik-Savinja Alps of Slovenia, which represent the southeastern-most extension of the Alpine topographic chain at the transition to the Dinarides (Fig.
Geographical position and river drainage systems of the Julian Alps and the Kamnik Savinja Alps range. Sampling sites: 1 Turjeva jama 2 Ložekarjeva jama 3 Konečka zijalka 4 Potočka zijalka 5 Jama na Zgornjih Brsnikih 6 Tomažičeva zijalka 7 Kamniška jama 8 Ihanščica cave. Digital terrain model from
Overview of cave locality information. For each of the seven cave localities the investigated specimens, their museum ID, latitude and longitude data, date of collection, elevation and a measurement of the ambient cave temperature are given. Leg. Rajko Slapnik. Museum ID’s refer to CSR SASA, RS, or SMNH collections.
Cave locality | Specimen ID (museum ID) | Lat. | Long. | Date | Elevation [m] | air T [°C] |
---|---|---|---|---|---|---|
Turjeva jama, Robič, Kobarid, Slovenia (region locus typicus) | 1 (37013) | 46.2435 | 13.5046 | 19.10.2007 | 253 | 9.3 (on 01.06.2013) |
2 (37013a) | 19.10.2007 | |||||
3 (RS0063a) | 01.06.2013 | |||||
Ložekarjeva jama, Olševa mountain, Kamnik-Savinja Alps, Slovenia | 4 (38474a) (MC SMNH 3291) |
46.4268 | 14.6240 | 28.09.2009 | 1050 | 7.9 (on 28.09.2009) |
5 (38474a) | 28.09.2009 | |||||
Konečka zijalka, Šmihel nad Mozirjem, Mozirje, Kamnik-Savinja Alps, Slovenia | 6 (37698a) (21675) |
46.4024 | 14.9393 | 16.08.2008 | 820 | 7.5 (on 16.08.2008) |
7 (37698a) | 16.08.2008 | |||||
8 (37698a) | 16.08.2008 | |||||
Potočka zijalka, Olševa mountain, Kamnik-Savinja Alps, Slovenia | 9 (40600a-1) (RS0059) |
46.4493 | 14.6693 | 08.06.2012 | 1630 | 5.5 (on 08.06.2012) |
10 (40600a-2) | 08.06.2012 | |||||
Jama na Zgornjih Brsnikih (= Jama pod Farjevim plazom, Jama pod Mokrico), Mokrica mountain, Kamniška Bistrica, Kamnik-Savinja Alps, Slovenia | 11 (39948a) (MC SMNH 2216) |
46.3093 | 14.5832 | 05.06.2001 | 980 | 6.0–10.2 (in 1997) |
12 (39948a) | 05.06.2001 | |||||
13 (39948a) | 05.06.2001 | |||||
Tomažičeva zijalka, Podvolovljek, Kamnik-Savinja Alps, Slovenia (locus typicus) | 14 (40596a-1) | 46.3134 | 14.6982 | 30.04.2012 | 590 | 8.9 (on 30.04.2012) |
15 (40596a-2) | 30.04.2012 | |||||
Kamniška jama, Zeleniške Špice, Kamniška Bistrica, Kamnik-Savinja Alps, Slovenia | 16 (40595a-1) | 46.3386 | 14.6124 | 01.05.2012 | 1400 | 5.1–6.1 (between 01.05.2003–01.05.2004) |
Ihanščica cave, Ihan, Ljubljana, Slovenia | 30014, 30042 (RS0103; RS0104) |
46.1216 | 14.6476 | 17.5.1969 11.10.2013 |
415 | Not recorded |
Geologically, the Slovenian Alps belong to the Southern Alpine thrust-belt, which formed during the last 20 MY and is still tectonically active, as evidenced by considerable seismic activity (Fig.
Overview of morphological assignments, molecular species delimitation results and integrative taxonomic treatment of each of the four clusters, which are indicated by the longitudinal black bars for either the ABDG or the SP approach. Numbers indicate individual specimens; ABGD = Automatic Barcode Gap Discovery; SP = Statistical Parsimony.
Since historical species designations are geographically very general (i.e. “Carniola” or “Krain” for the Eastern Alpine region including southern Austria and Slovenia) and taxonomic data was recorded during eras of former political boundaries, geographical designations and taxonomic traditions, we have endeavored to collect live material of Z. isselianum and allied species as close to where the initial sites of species descriptions may have been. We remark however, that although the subspecies, Zospeum alpestre likanum Bole, 1960 could be considered in a broader scope of this investigation, it’s geographic type locality (Gornja Cerovačka pećina, Gračac, Croatia) is located distant from the core, proximal Eastern Alpine sites comprising this study (Fig.
Overview of the morphological and genetic results. The 16 specimens (#) can be attributed to five morphospecies. The genetic delimitation reveals four clusters (color-coded), with four lineages (L1−L4). Individual BOLD-ID. (http://www.boldsystems.org/) of each specimen and a link to the broader study of
Morphospecies | # | Lineage | BOLD-ID | Comments |
---|---|---|---|---|
Z. isselianum Pollonera, 1887 | 1 | L1 | BARCA121-10 | same as Z6 from |
2 | L1 | BARCA213-15* | same as Z6 from |
|
3 | L1 | BARCA214-15* | same as Z6 from |
|
11 | L3 | BARCA122-10 | same as Z5 from |
|
12 | L3 | BARCA216-15* | same as Z5 from |
|
13 | L3 | BARCA217-15* | same as Z5 from |
|
6 | L2 | BARCA123-10 | same as Z7 from |
|
7 | L2 | BARCA124-10 | same as Z7 from |
|
8 | L2 | BARCA215-15* | same as Z7 from |
|
Z. cf. amoenum | 9 | L2 | BARCA211-13* | same as Z7 from |
10 | L2 | BARCA212-13* | same as Z7 from |
|
Z. alpestre alpestre | 16 | L3 | BARCA218-15* | same as Z5 from |
Z. alpestre bolei | 14 | L3 | BARCA219-15* | same as Z5 from |
15 | L3 | BARCA220-15* | same as Z5 from |
|
Z. alpestre kupitzense | 4 | L4 | BARCA125-10 | same as Z1 from |
5 | L4 | BARCA126-10 | same as Z1 from |
The barcoding region of the cytochrome c oxidase subunit I (COI) gene was amplified for 16 specimens from 7 caves including the morphospecies Z. isselianum (9 specimens, 3 caves), Zospeum cf. amoenum (Frauenfeld, 1856) (2 specimens, 1 cave), Z. alpestre bolei (2 specimens, 1 cave), Z. alpestre alpestre (1 specimen, 1 cave) and Z. alpestre kupitzense (2 specimens, 1 cave) (Table
COI sequences for genetic delimitation were either retrieved from the BOLD project “BARCA” or generated anew within this study (Tab.
The Automatic Barcode Gap Discovery (ABGD) procedure was conducted (
To differentiate and compare the geographically proximal species comprising our study, available technology such as X-ray-nanotomography (nano-CT) and SEM are used to access new information from traditional morphological characters (external morphology, radula and sinuosity of the columellar lamella). The position and the degree of sinuosity of the columellar lamella is a widely accepted diagnostic character within the Carychiidae (
Shells of three populations of Zospeum isselianum from three distinct genetic clusters (see results; L1, L2 and L3) were measured according to
Zospeum isselianum conspecifics from neotype locality (Turjeva jama), CSR SASA 37013/01−10, Turjeva jama, Robič, Kobarid, Slovenia. SH Shell height. SW Shell width. PH Peristome height. PD Peristome diameter. Lcol Contact between the columellar edge of peristome and last whorl, all in mm.
SH | SW | PH | PD | Lcol | SW/SH | AW/AH | Lcol/SH | |
---|---|---|---|---|---|---|---|---|
37013/01 | 1.05 | 0.91 | 0.70 | 0.47 | 0.34 | 0.865 | 0.676 | 0.321 |
37013/02 | 1.35 | 1.02 | 0.71 | 0.58 | 0.46 | 0.755 | 0.813 | 0.341 |
37013/03 | 1.44 | 0.96 | 0.76 | 0.62 | 0.49 | 0.670 | 0.811 | 0.344 |
37013/04 | 1.36 | 0.95 | 0.69 | 0.53 | 0.54 | 0.698 | 0.766 | 0.396 |
37013/05 | 1.30 | 0.97 | 0.77 | 0.60 | 0.58 | 0.744 | 0.775 | 0.444 |
37013/06 | 1.39 | 0.95 | 0.70 | 0.58 | 0.52 | 0.686 | 0.825 | 0.377 |
37013/07 | 1.36 | 0.98 | 0.68 | 0.57 | 0.44 | 0.721 | 0.842 | 0.323 |
37013/08 | 1.37 | 0.92 | 0.68 | 0.60 | 0.54 | 0.673 | 0.893 | 0.396 |
37013/09 | 1.41 | 1.02 | 0.72 | 0.61 | 0.53 | 0.726 | 0.847 | 0.375 |
37013/10 | 1.31 | 0.96 | 0.68 | 0.56 | 0.55 | 0.737 | 0.813 | 0.423 |
Shell dimensions. Mean, maximum (max), minimum (min), and standard deviation (sd) of shell measurements of 3 populations of Zospeum isselianum, Turjeva jama, N=40; Jama na Zgornjih Brsnikih N=40 and Zospeum sp. Konečka zijalka, N=32. SH Shell height. SW Shell width. PH Peristome height. PD Peristome diameter. Lcol Contact between columellar edge of peristome and last whorl, all in mm.
SH | SW | PH | PD | Lcol | SW/SH | PH/PD | Lcol/SH | |
---|---|---|---|---|---|---|---|---|
Zospeum isselianum Turjeva jama | ||||||||
mean | 1.35 | 0.97 | 0.70 | 0.58 | 0.49 | 0.72 | 0.83 | 0.36 |
max | 1.63 | 1.09 | 0.84 | 0.62 | 0.59 | 0.86 | 0.92 | 0.45 |
min | 1.05 | 0.91 | 0.65 | 0.47 | 0.34 | 0.63 | 0.68 | 0.24 |
sd | 0.086 | 0.036 | 0.033 | 0.028 | 0.052 | 0.039 | 0.046 | 0.038 |
Zospeum isselianum Jama na Zgornjih Brsnikih | ||||||||
mean | 1.53 | 1.07 | 0.75 | 0.60 | 0.41 | 0.70 | 0.80 | 0.27 |
max | 1.63 | 1.12 | 0.93 | 0.87 | 0.58 | 0.76 | 0.93 | 0.39 |
min | 1.38 | 0.87 | 0.67 | 0.54 | 0.29 | 0.55 | 0.68 | 0.18 |
sd | 0.051 | 0.055 | 0.044 | 0.053 | 0.060 | 0.037 | 0.048 | 0.0417 |
Zospeum isselianum Konečka zijalka | ||||||||
mean | 1.37 | 1.02 | 0.69 | 0.61 | 0.53 | 0.75 | 0.87 | 0.35 |
max | 1.60 | 1.25 | 0.80 | 0.74 | 0.67 | 0.84 | 1.06 | 0.40 |
min | 1.19 | 0.93 | 0.58 | 0.51 | 0.42 | 0.68 | 0.76 | 0.30 |
sd | 0.084 | 0.066 | 0.051 | 0.039 | 0.066 | 0.037 | 0.055 | 0.053 |
In order to assess historical species hypotheses in context here, access to type material housed in museum collections is paramount. Type material whenever possible has been obtained for this study (Figs
The formaldehyde-preserved specimens of conchologically determined (RS) Zospeum isselianum (Konečka zijalka) and Z. isselianum (Turjeva jama, neotype locality) were dissolved in a solution of 5% acetic acid and 10% formaldehyde to dissolve the shell. The snails were subsequently dehydrated in an increasing ethanol series. Pre-infiltration followed with a 96% ethanol and base liquid solution of Technovit 7100 (50:50 mixture) for two hours and then infiltrated over night following the manufacturer’s instructions (Technovit 7100, Heraeus Kulzer GmbH, Wehrheim/Ts., Germany). The specimens were subsequently embedded in cold-polymerizing hydroxyethyl methacrylate resin. Serial sections (1 µm) were prepared using the Leica RM2165 automatic Rotation Microtome (Leica Microsystems Nussloch, Germany), stained with toluidine blue and examined with a high-powered microscope (Leica DM LB2). Histological photomicrographs were taken using a digital camera (Leica DC 300F) and further processed using the software Photoshop 6.0 (Adobe Systems Inc., San Jose, CA, USA).
The radula of Zospeum isselianum (Turjeva jama, neotype locality) was prepared according to
Shells or preserved full-bodied snails selected for SEM were microscopically evaluated for stability (presence of cracks, shell thickness, evidence of erosion). The sturdiest shells were first wetted in a dish of 80% ethanol or water and then manually brushed clean of cave encrustations using fine, tapered dental brushes, whereby each specimen was gently rotated back and forth between the brushes until it was sediment free. Specimens were then mounted onto double-sided carbon tabs. The samples were sputtered and processed using the same systems as for the radula.
Specimens were imaged using a nano-computed tomography system (nano-CT), manufactured and developed by Bruker-Micro-CT/SkyScan (SkyScan 2011, Kontich, Belgium) at the Department of Experimental Radiology, Justus-Liebig University Biomedical Research Center Seltersberg (BFS), Giessen, Germany. The system contains an open pumped type X-ray source, a LaB6 cathode and a transmission anode consisting of a tungsten-coated beryllium window. Enhanced edge sharpness and submicron resolution are gained by a high-focused X-ray spot of <400 nm side length (see
Zospeum species were photographed using a Kontron-Electronik-ProgRes-3012 microscope camera (Jena, Germany) and a Leitz MZ12 stereomicroscope.
The ABGD approach delineates four clusters (Fig.
Based on the four revealed evolutionary lineages, evidence for delineation using non-molecular methods encompasses four taxonomic consequences:
Z. isselianum Pollonera, 1887 (L1) is defined from the area of its new precise type locality (Turjeva jama, Robič, Kobarid, Slovenia) whereby a neotype is designated and a taxonomic re-description is provided.
We recognize a highly morphologically variable Zospeum sp. (L2) (Konečka zijalka) with conchological affinity to Z. isselianum and Z. cf. amoenum. However, Z. amoenum topotypic material is necessary before species status can be clarified. Histological examination of L2 provides novel insights into the anatomy of Zospeum in a comparative framework with other Ellobioidea.
Z. alpestre (L3) encompasses subspecies comprising the Z. alpestre complex, including Z. alpestre alpestre and Zospeum alpestre bolei plus three specimens of Z. isselianum.
Z. kupitzense (L4) is elevated from subspecies rank (Z. alpestre kupitzense) based on a clear differentiation of columellar configuration and lamellar extension outside the aperture.
Zospeum alpestre —
Zospeum isselianum Pollonera, 1887
Zospeum alpestre —
Zospeum alpestre —
The two syntypes of Zospeum isselianum were collected in debris of the Natisone River in the northeastern Italian region of Friuli (
“This species just resembles Z. obesum Schmidt, Z. alpestre Freyer (pars), and Z. nyctozoilum Bourg. From the first it differs because it lacks an obsolete columellar fold, because it has the umbilicus more open, the peristome less swollen, and the apex not acute. From the second it differs because it has the umbilicus less cramped, the apex less acute (indeed mammillatus), the sutures deeper and it has the last lap with a lower development. From the third it differs because the right margin is a little arched (not reflected) and it has the umbilicus more open (Issel)” (
Neotype (CSR SASA 37013/37013a) (Fig.
Shell minute, transparent when fresh, conical with entire, half-roundish and more or less thickened peristome; parietal lamella; columella non-introrse, secondary columellar dilatation just above umbilical indentation; columellar lamella inclinate.
Measurements of neotype specimen CSR SASA 37013 (Fig.
Measurements of conspecifics from Turjeva jama are provided in Table
(Figs
Although we question past distribution records (Fig.
Live Z. isselianum were found in Turjeva jama on mud and muddy walls at the end of the cave and adjacent to the central pit located in the middle of the cave. Bats were seen in the vicinity of the collection site.
In the caves sampled for this study, empty shells were sparsely found in sediment and live individuals sparsely populated certain cave walls during the summer season. These findings suggest that Z. isselianum occurred there for more than one season, and that these populations are/were not immediately threatened. Still, on a global scale, its distribution is fragmented and likely limited to far less than 312 caves within a radius of 20,000 km2. In conjunction with the categories for the IUCN Red List (
This species shows a wide range of variability in the shell. The parietal lamella with its nondescript shape and configuration has been considered characteristic (
As known for pulmonates, the anterior digestive system (Fig.
Light micrograph showing histological overview of the digestive system of Zospeum sp. (Konečka zijalka) (CSR SASA 21675). A Mouth and buccal apparatus. Mouth slit between oral lappets (m), crescent-shaped jaw (j), radula (r), pharynx (ph), digestive gland (dg), intestine (i), kidney (k), columellar muscle (cm), and pedal ganglia (peg)BMantle gland (mag), sigmoid intestine (si), radular sac (rs), cerebral ganglion (cg), mucus glands (mg), kidney (k), heart showing auricle and ventricle (h), digestive gland (dg), columellar muscle (cm), oesophagus (oe) and salivary glands (sg).
A Light micrograph showing the histological appearance of Zospeum sp. (Konečka zijalka) (CSR SASA 21675) with the mantel cavity (mc), mucus glands (mg), oesophagus (oe) bearing thick cuticular sheath (cs) and leading into the crop (c), the buccal ganglia (bg), and the cephalopodial portion of the reproductive system including penis (p) and vas deferens (vd)B Paired cerebral ganglia united by the cerebral commissure situated just dorsal to the origin of the oesophagus in the buccal mass.
Images of the radula of Zospeum isselianum (Turjeva jama) (CSR SASA 37013 conspecific) and Zospeum sp. (Konečka zijalka) (CSR SASA 21675). A−DSEM of the radular ribbon of topotypic Z. isselianum A Radular ribbon showing adhesive layer of contact zone with odontophore BSEM overview showing rachidian teeth (r) and lateral teeth (l)C Rows of lateral teeth bearing fine medial groove D Marginal teeth E Light micrograph showing the histological appearance of the radular sheath of Zospeum sp. (Konečka zijalka) showing the inferior epithelium (ie) attached to the radular membrane (rm)mineralizing cells (mc) with degenerating nuclei and radular teeth (rt).
Light micrograph showing histological appearance of the radular complex of Zospeumsp. (Konečka zijalka) (CSR SASA 21675). A Odontoblasts (odb) grouped in lower posterior section of radular sheath, radular teeth (rt), odontophore (od), and collostyle (col)B Section through an acinus of the ovotestis showing some stages of development of sustentacular cells (Sertoli cells) (sc) with spermatogonia (spg), spermatids (sp) and oogonia (og).
Light micrograph of the histological appearance of the digestive tract of Zospeum sp. (Konečka zijalka) (CSR SASA 21675). A−B Bilobed muscular gizzard (mg) with epithelium showing columnar cells (cc) with microvillous apical surfaces (mv), posterior loop of the intestine (pi) with ciliated epithelium (ce)B Elaborate muscular plates (mp), thick cuticle (cu), highly ciliated rectum (r), gastric pouch (gp), digestive gland (dg) and hermaphroditic duct.
Light micrograph of a section of the reproductive tract of Zospeum sp. (Konečka zijalka) (CSR SASA 21675). AGonad (g) with acinus of the ovotestis including the germinal epithelial ring (ger) surrounding the acinar lumina (al), sustentacular cells (Sertoli cells) (sc) and oogonia (og)BDigestive gland (dg), hermaphroditic duct (hd) lined with cilia (c), posterior loop of the intestine (pi), receptaculum seminis (rs) containing allospermatozoa with spermatozoa oriented towards and apical ends touching the epithelial cells, and long motile cilia (ce) of intestine (i).
The long intestine of Zospeum is visible from the outside of the shell when the snail is alive. The intestine encompasses an anteriorly directed loop frequently seen amidst other members of the Ellobioidea (
Though not part of the digestive system but nonetheless encompassed in the broader scope of these slides, the mantle gland (mg) (Fig.
The Carychiidae are considered to possess the simplest ellobioid radula (
Each column of teeth is formed by a single set of a number of odontoblasts (odb) located at the lower posterior end of the radular sheath as seen here for Zospeum sp. (Konečka zijalka) (Fig.
One of the most prominent aspects of the cephalopedal region of Zospeum sp. (Konečka zijalka) here is the retracted conical penis and the long vas deferens embedded in the body wall, running parallel to but independent of the penis (Fig.
The orange-yellowish gonad (when fresh) is situated between the uppermost lobes of the digestive gland in the hollow, apical whorls of the teleoconch (Fig.
The CNS of Zospeum sp. (Konečka zijalka) follows that of
Z. isselianum (RS0103) from Ihanščica cave (Figs
The robust, broadly clavate columella of Zospeum alpestre of Kamniška jama (Fig.
Zospeum kupitzense (Figs
The umbilical notch of Z. isselianum (Turjeva jama, neotype locality) (Fig.
The deep interconnected pits forming continuous rows of wavy pattern on the upper teleoconch are markedly more pronounced in topotypic Z. isselianum (Turjeva jama) (Fig.
Our molecular delimitation results singularly imply that:
Zospeum isselianum (L1) is validated by molecular data from the area of the new precise type locality (Turjeva jama, Robič, Kobarid, Slovenia).
Zospeum sp. (Konečka zijalka) (L2) is an unknown, highly morphologically variable Zospeum lineage (comprising specimens of Z. isselianum and Z. cf. amoenum) with ambiguous identity.
Polytypy within the species Z. alpestre (i.e. Z. alpestre alpestre, Zospeum alpestre kupitzense and Zospeum alpestre bolei) needs reassessment.
Our studies of Zospeum shell morphology of the allied Eastern Alpine species show the wide degree of external shell variability and evidence of convergent evolution within these taxa. These investigations corroborate the findings of
The age of the shell, superficial encrustations, degree of preservation/freshness, and the degree of biomineralization affecting shell hardness vary, and thus, generally influenced moderate forms of superficial shell texture.
Zospeum shows characteristic pulmonate design with ellobioid affinity. The gross anatomy of Zospeum (Konečka zijalka) does not deviate significantly from other investigated Zospeum species (
The radulae of Zospeum isselianum (Turjeva jama) here and Zospeum sp. (Konečka zijalka) (
Based on the most unambiguous shell character found in this study, namely, the configuration of the columella and independently, the relationship of the columellar lamella with it, the bigger morphological assessment-vs-molecular delimitation picture can be broken down as follows:
Columella slender, twisted; secondary columellar dilatation at base of columella; columellar lamella inclinate, extended. Lineage 1: Zospeum isselianum from new type locality.
Columella robust, cylindrical, smooth; no columellar lamella: Z. amoenum (Potočka zijalka).
Columella robust, hint of secondary dilation at base, smooth; reduced, un-elaborated (i.e. non-extended) columellar lamella. Lineage 2: Zospeum sp. (Konečka zijalka) appears to be morphologically an intermediary form of Z. amoenum and Z. isselianum morphotypes.
Columella broadly clavate; columellar lamella thin and finely sinuous, extending to a short horizontal plane within the penultimate whorl. Lineage 3: Z. alpestre (Kamniška jama).
Columella slender, twisted; slight secondary columellar dilation at base of columella; columellar lamella only similar to Z. alpestre but not the columella; pronounced parietal lamella formed from the inner track of the columellar lamella; parietal lamella remarkably long, extending to middle of shell above the umbilicus (bottom perspective). Lineage 4: Z. kupitzense.
This morphological analysis, in conjunction with the molecular results, suggests that lineage 2 Zospeum sp. (Konečka zijalka) possesses an intermediary columellar form and shows morphological affinity to both Z. amoenum and Z. isselianum. We cannot designate species status for this material at this time. On the one hand, these individuals are not Z. isselianum and can thus, be excluded as this species by our study here. On the other hand, Z. amoenum cannot be excluded because we did not investigate type-material of this species. This means the examination of L2 should be the focus of another investigation, incorporating topotypic material of Z. amoenum (locus typicus: Velika Pasjica, Gornji Ig, Slovenia) to clarify this issue. Interestingly, this cave is also the type locality of Zospeum spelaeum schmidtii. In regards to the bigger picture here, it is remarkable that
Based on the configuration of the columella plus the independent configuration of the columellar lamella, morphological differentiation of these lineages was generally possible. Although aperture dimensions and the expression of the columellar lamella in the aperture were highly rated in earlier assessments of Zospeum (
Four evolutionary lineages of Eastern Alpine Zospeum were clearly molecularly delineated and assessed using various non-molecular methods. Based on differentiating shell features and the molecular species delimitation, a neotype for Zospeum isselianum was described. This study performed the first, non-destructive 3D-reconstruction using X-ray tomography (nano-CT) to differentiate shells of conchologically and geographically allied species of Zospeum. Since external shell morphology is too variable, specific focus was given to internal characters such as to the configuration of the columella and the orientation of the columellar lamella within the ovate conical shell. The columella was found to be the most reliable conchological character differentiating species of Eastern Alpine Zospeum taxa. SEM assessment infers that a median groove incised in the middle cusp of the lateral teeth of Z. isselianum (Turjeva jama, neotype locality) here and Zospeum sp. (Konečka zijalka) of previous investigations (
Special gratitude goes to Massimo Prodan & Floriana Umani (Trieste) for their kind and expert assistance in translation of the primary literature and to Iztok Sajko for drafting the distribution map. We thank Elena Gavetti (MRSN) for providing valuable primary literature and imaging the Pollonera type material. We acknowledge Maria Tavano’s (MSNG) help in accessing the Arturo Issel collection. Appreciation also goes to Willy de Mattia for his insightful discussions and help with translating his earlier work from the Italian literature. We are grateful to Ruud Bank for his constructive input and help in literature acquisition. We thank Ronald Janssen (SMF), Anita Eschner (NHMW), Matjaž Kuntner (CSR SASA) and Tomi Trilar (SMNH) for providing access to essential type material. Much gratitude also goes to Sigrid Hof (SMF), Katherina Jaksch (NHMW) and Emmanuel Tardy (MHNG) for their help and patience in imaging these often, “uncooperative” shells. We especially thank Manfred Ruppel (Goethe University, Frankfurt) for his insights and generous time investment at the SEM. Gratitude also goes to Gabriele Elter (Goethe University, Frankfurt) for her help with sectioning the material from Turjeva jama. Special thanks also go to Eike Neubert (NMBE) and the two reviewers, Edi Gittenberger and Roman Ozimec for their helpful comments towards improving the manuscript. We acknowledge the speleological assistance from Jana Valentinčič in our team efforts to collect material. This work was made possible in part by a grant from The Malacological Society of London for SEM resources. This research received support from the SYNTHESYS Project http://www.synthesys.info/, which is financed by the European Community Research Infrastructure Action under the FP7 “Capacities” Program.
Major funding for this project was provided by the research funding program ‘LOEWE’ –Landes-Offensive zur Entwicklung Wissentschaftlich-ökonomischer Exzellenz’ of the Ministry of Higher Education, Research, and the Arts for the State of Hessen. We also thank the Ministry of Higher Education, Science and Technology of the Slovene Republic (P1-0236 Biodiversity and Gradients) for their continued support of our research.
List of localities of Z. isselianum
Data type: occurrence
Explanation note: List of localities of Z. isselianum. A − Austria, BiH − Bosnia and Herzegovina, Cro − Croatia, Slo − Slovenia, Ita − Italy, CBSS − Collection of Croatian Biospeleological Society, CSR SASA – Malacological collection of the Biological Institute of the Centre for Scientific Research of the Slovenian Academy of Sciences and Arts in Ljubljana, SMNH – Malacological collection of the Slovenian Museum of Natural History, LIT – data from literature.