New Zospeum species (Gastropoda, Ellobioidea, Carychiidae) from 980 m depth in the Lukina Jama – Trojama cave system (Velebit Mts., Croatia)

A new species of the eutroglobiont gastropod taxon Zospeum Bourguignat, 1856 is described. Zospeum tholussum sp. n. is characterized based on a population from the Lukina Jama–Trojama cave system (Ve­ lebit Mts., Croatia). A single living specimen occurred at 980 m depth. The species is morphologically related to Zospeum amoenum (Frauenfeld, 1856), but can be readily distinguished from the latter by the presence of a weak columellar fold and its dome­like structured 2nd whorl. DNA barcoding is capable to clearly delineate Z. tholussum from other Zospeum spp. as well.

Their taxonomic history has been shaped by confusing and sometimes contradicting (sub)species assignments, primarily as a result of the general problem to distinguish intraspecific and interspecific shell variability (Bole 1974), which for decades was the main source of taxonomic information.Recently, Weigand et al. (2011) implement ed DNA barcodes to molecularly identify species (i.e.DNA barcoding) based on a fragment of the mitochondrialencoded (mtDNA) Cytochrome C Oxidase Subunit I (COI) locus (Hajibabaei et al. 2007).In a subsequent study and by addressing the evolutionary history of Carychiidae, Weigand et al. (2013) revealed several morpho logically unrecognized (cryptic) Zospeum species and demonstrated the suitability of molecular data for taxonomic purposes in this group.All known Zospeum have lost visual orientation and are considered true eutroglobi onts (see Sket 2008).Information on their biology is scarce, but species seem to possess a limited active movement (Slapnik 2001) and to prefer muddy and permanently wet subterranean microhabitats (Jochum et al. 2012), as generally found along the drain age system of a cave.Weigand et al. (2013) proposed that dispersal may primarily oc cur via passive transportation (e.g. by water or larger mammals).
Here, a new Zospeum population from the Lukina Jama-Trojama cave system situ ated in the Velebit mountain range of Croatia is characterized (Fig. 1).On the basis of molecular and conchological data, a new species from 980 m depth-Zospeum tholussum Weigand, sp.n.-is described.

Conchological measurements
Specimen shells were measured by taking individual images with 456 pixels corre sponding to 1 mm.The total shell height (SH), shell width (SW), aperture height (AH), aperture width (AW) and the number of whorls were measured.Moreover, ra tios for SH/SW and AH/AW were calculated.

Non-invasive DNA isolation, PCR and sequencing
Specimen shells of livecollected Zospeum tholussum sp.n. (single specimen) and Zospeum amoenum (Frauenfeld, 1856) (two specimens) were kept intact by using a protocol for a noninvasive DNA isolation method originally described in Schizas et al. (1997) and partially modified after BöttgerSchnack and Machida (2011).Each specimen was cleaned and then dried in a 0.2 mL PCRtube by raising the temperature once up to 94 °C.A volume of 9 µL ddH 2 O and 1 µL 10x PCRbuffer were added.The mixture was heated for 2 min at 94 °C; 1.3 µL Proteinase K were added and the solu tion homogenized by gentle mixing.An incubation step was performed for 15 min at 55 °C followed by 10 min at 70 °C.The incubation was repeated once.10 µL of Gene Releaser (Bioventures Inc.) were added and the following thermocycler protocol used: 30 s at 65 °C, 30 s at 8 °C, 1.5 min at 65 °C, 3 min at 97 °C, 1 min at 8 °C, 3 min at 65 °C, 1 min at 97 °C, 1 min at 65 °C, 5 min at 80 °C and storage at 4 °C.The reaction mixture including the intact shell was centrifuged for 1 min using a table centrifuge.The clean phase containing the DNA was collected and transferred to another 0.2 mL PCRtube.The shell was cleaned from the remains of the Gene Releaser chemicals by alternate rinsing in water and ethanol.10-15 µL of AE Buffer (DNeasy Kit, Qiagen) were added to the isolated DNA.
PCR conditions were the same as described in Weigand et al. (2011), sequencing was performed at Source BioScience (LifeSciences, Berlin, Germany) following the companies instructions.

DNA barcoding
DNA barcodes, chromatograms, images, geographic data and further information of the genetically analysed Zospeum tholussum (BOLDID BARCA21013) and Z. amoenum (BOLDIDs BARCA21113 and BARCA21213) specimens are deposited in the Barcode of Life Database (BOLD) (Ratnasingham and Hebert 2007) in the project "Barcoding Carychiid Microsnails" [BARCA].The obtained COIsequences were compared with other Zospeum species in BOLD.
Diagnosis.The general shape of Zospeum tholussum sp.n. resembles larger speci mens of Zospeum amoenum (Frauenfeld, 1856) from which it can be best delineated by the domelike structured 2 nd whorl (Fig. 2), the weak but present columellar fold (Fig. 3) and by means of their DNA barcodes (11.7 %-12.1 % genetic pdistance between both species).
Habitat.The single living specimen was found in an unnamed large chamber at 980 m depth (85 m long, 70 m wide) with lots of stones, rocks and sand (Fig. 1, right site).A temporal small stream of running water was present close to the collecting site.Air temperature was between 3.3-3.5 °C (depending on the measurement device), Table 1.Conchological measurements for Zospeum tholussum.Seven paratypes (Ind.1-7) and the holo type specimen (Ind.8) were measured.All measurements are given in millimeter.Additionally, ratios of shell height / shell width (sh/sw) and aperture height / aperture width (ah/aw) were calculated.The range of variability is given for all parameters.water temperature 5.1 °C and air humidity 100 %.Shells were observed beginning from 800 m depth till the bottom of the cave.Shells were generally found on layers of mud (Fig. 1, left site).The first 200 m of the entrance passage of Lukina Jama are permanently covered by varying levels of snow and ice.

Ind
Etymology.The Latin word tholus means dome or cupola and refers to the re markable domelike shape of the 2 nd whorl.
Remarks.In addition to the newly described Zospeum tholussum, a second Zospeum species is present in the Lukina Jama-Trojama cave system (Fig. 4C).This spe cies can be differentiated from Z. tholussum by the presence of a tooth, its general shell shape, more prominent columellar fold and absence of the characteristic domelike structured 2 nd whorl.Because only shells were found, no DNA barcodes are available for this species.
So far, Zospeum tholussum is only known from the Lukina Jama-Trojama cave system.However, this cave system is situated in the distribution range of the morpho logically related species Zospeum amoenum (Frauenfeld, 1856), which inhabits caves of the West Balkan of North Slovenia, West Croatia, Bosnia and Herzegovina and South to Montenegro (Bole 1974).Absolon (1916) named but did not describe an other morphologically related species Zospeum troglobalcanicum.Since Weigand et al. (2013) revealed that Zospeum spp. with an inferred large distribution range are particularly prone to host additional, morphologically unrecognized species, some of the distribution records for Z. amoenum may very well refer to Z. tholussum.Moreo ver, the intraspecific genetic and conchological variability of the new species should be investigated in greater detail, thus to judge about the taxonomic information content of the diagnostic characters applied within this description.
The preliminary information on the habitat of Zospeum tholussum is in congruence with previous findings for Zospeum.Interestingly, the grazinglabyrinthlike structure, in which most of the shells were embedded (Fig. 1, left site), has been already observed during caving expeditions in Northern Spain (Jochum et al. 2012).Finally, the pres ence of a temporal stream at the collecting site of the living specimen further supports a passive dispersal scenario as suggested by Weigand et al. (2013).

Figure 1 .
Figure 1.The Lukina Jama-Trojama cave system.Overview of the geographical position and 3D cave crosssection.In the latter, the region of collected shells (1) and the collection site of the living specimen of Zospeum tholussum (2) are indicated.The 3D crosssection was provided by D. Bakšić et al. (2010), Croatian Speleological Server, http://www.speleologija.hr/lukinajama.Photos were taken by J. Bedek.

Figure 2 .
Figure 2. Holotype and paratypes of Zospeum tholussum.The holotype (former living specimen) is marked with a solid line; five paratype specimens (shells) are surrounded by dotted lines.

Figure 3 .
Figure 3. Columellar fold views of Zospeum tholussum.The columellar fold of the broken paratype specimen shell is shown in clockwise rotation.