Testate Amoebae in Karst Caves of the Dinaric Arc (South-Eastern Europe) with a Description of Centropyxis bipilata sp. nov. Author Baković, Najla Author Siemensma, Ferry J. Author Baković, Robert Author Rubinić, Josip text Acta Protozoologica 2019 58 4 201 215 http://dx.doi.org/10.4467/16890027ap.19.018.12020 journal article 10.4467/16890027AP.19.018.12020 1689-0027 12637219 Centropyxis bipilata sp. nov. ( Fig. 5 ) urn:lsid:zoobank.org:act: BFDF2F53-30E3-4450- B88B-F8EBB7DB85D5 Description : In dorsal view, the test is rounded and usually slightly wider than long; in lateral view, the height of the test is more or less half (0.4–0.6) of the diameter of the test. The dorsal part gradually lowers towards the anterior side, the ventral surface is flattened. The test is built of granular organic cement, yellowish to brown, more or less embedded with small mineral particles. Some tests lack these xenosomes, others are more densely covered. The test never bears spines. The aperture is eccentric, invaginated and ovoid, elliptical or circular. The distance from the centre of the aperture to the anterior edge is approximately 0.4 (0.37–0.46) of the test length. The edge of the pseudostome curves inward, creating a small but clear ring that is usually slightly darker than the rest of the test. The anterior part of the edge is higher and always connected to the inner anterior-dorsal wall by two struts. Between those struts there is a more or less U-shaped opening, the size of which is variable per specimen, usually large, but sometimes smaller ( Fig. 5 ). Lobopodia as common for this genus. Nucleus not observed. Dimensions : Length of the test 52–81 µm (61.7 µm); width 55–75 µm (63.0 µm); height 28–43 µm; diameter of the pseudostome 12–22 × 11–25 µm (17.2 × 17.8 µm) (n = 29). Ecology : Centropyxis bipilata sp. nov. inhabits sediments in aquatic and terrestrial habitats and transitional habitats in freshwater karst caves in Croatia and Bosnia and Herzegovina . It is common in habitat whose energy input is dependent on the seeping water from the surface, on exogenous flooding and on the habitats that where additionally enriched with bats guano. All habitats, where this species was found, are out of the reach of the sunlight during the whole year. The record- ed temperature on habitats of this species ranges from 18°C (e.g. Topla peć) to 4.1°C (e.g. Veternica cave). Table 2. Biodiversity of testate amoebae in all sampled caves

Species	Caves where species was observed
Arcella sp.	STR
Assulina muscorum Greeff, 1888	LIS, TAM
Centropyxis aerophila Deflandre, 1929	DAH, JOP, KOV, LIS, LTV STR, TAM
Centropyxis constricta (Ehrenberg, 1841) Penard, 1890 .	SIN, TAM
Centropyxis elongata (Penard, 1890) Thomas, 1959	BAR, KOV, TOP
Centropyxis plagiostoma Bonnet & Thomas, 1955	TAM, VEP
Centropyxis bipilata sp. nov.	BIL, DAH, CRV, ČAK, HOR, HUK, KOT, KOV, LIS, LTV, MLJ, PRA, ROG, TAM, TNJ, TOP, VEP, VET
Centropyxis spp.	DAH, LIS, SUV
cf. Conicocassis sp.	BUK, LIS
Cryptodifflugia oviformis Penard, 1902	BIL, DAH, JOP, KOV, LTV, STR, SUV, TAM, TNJ, TOP, VEP
Cyclopyxis eurystoma Deflandre, 1929	BAR, KOV, TAM, TOP
Cyclopyxis spp.	BAR, DAH, KOV, LIS, SIN, STR
Cyphoderia ampulla Ehrenberg, 1840	BAR, BUK, DAH, JOP, KOV, LTV
Difflugia cf. brevicolla Cash & Hopkinson, 1909	KOV
Difflugia lithophila Penard, 1902	KOV
Difflugia oblonga Ehrenberg, 1838	BAR, KOV
Difflugia pristis Penard, 1902	TAM
Difflugia pyriformis Perty, 1849	BAR, KOV, VEP
Difflugia spp.	BUK, JOP, KOV, VEP
Diplochlamys sp.	KOV
Euglypha bryophila Brown, 1911	BUK, KOV
Euglypha cf. laevis (Ehrenberg, 1832) Perty, 1849 *	DAH, TAM
Euglypha cf. rotunda Wailes, 1915 *	BUK, DAH, JOP, KOV, SUV, TNJ, TOP
Euglypha sp.	BAR, TAM, TNJ, VEP
Frenzelina sp. Penard, 1902	TOP
Heleopera petricola Leidy, 1879	VEP
Heleopera sp.	JOP
Lacogromia sp.	KOV
Meisterfeldia cf. vanhoornei (Beyens et Chardez, 1986) Brobov, 2016	TOP
Microchlamys patella (Claparède et Lachmann, 1859) Cockerell, 1911	KOV, LTV, STR, VEP
Paramphitrema sp. **	DAH
Paraquadrula irregularis Wallich, 1863	JOP, LTV
Parmulina cyathus Penard, 1902	VEP
Plagiopyxis declivis Bonnet, 1955	JOP, KOV, STR, VEP
Plagiopyxis sp.	TAM
Pseudodifflugia sp.	TOP, VEP
Tracheleuglypha dentata Deflandre, 1928	DAH, TNJ, TOP
Trinema complanatum Penard, 1890	BAR, SUV
Trinema enchelys Ehrenberg, 1838	BAR, KOV, SUV, TAM, TNJ, TOP, VEP
Trinema lineare Penard, 1890	BAR, BIB, BUK, DAH, DOB, KOV, SIN, STR, SUV, TAM, TOP, VEP
Testacea spp.**	BAR, DAH, JOP, LIS, TNJ, SIN, TAM, TOP, VEP

*Species identification can only be confirmed by using SEM. **(Generic) identification can only be confirmed by living specimens. Fig. 2. Total number of detected testate amoebae per cave Fig. 3. Examples of species found during this research (A. Centropyxis elongata ; B. Lacogromia sp. ; C. Cyclopyxis sp. ; D–E. Paramphitrema sp. ; F. Diplochlamys sp. ; G–H. cf. Conicocassis sp. ; Scale bars B 100 µm, all other 20 µm) Fig. 4. Biodiversity of protozoa in various cave habitat types (TAM – testate amoebae, CIL – ciliates, NAM – naked amoebae, FLG – heterotrophic flagellates, HEL – heliozoans) Fig. 5. Centropyxis bipilata sp. nov. A–C. Ventral view, arrows point to the position of the struts; D, F. Frontal view, showing the two struts connected to the dorsal part; E. Side view, showing one strut. A, C–E stacked images. Scale bars: 20 µm. Etymology : bi [from Latin bis ] = two; pilata [from Latin pila ] = pillar; bipilata refers to the two struts inside the shell. Type locality : Topla peć, Golubić Žegarski , Croatia Topla peć is a karst cave formed within a canyon of the river Krupa near the settlement of Golubić Žegarski ( Croatia ). It is a cave of simple morphology. The entrance is 4 m high and 4 m wide and exposed to sunlight from the south. The total length of the cave is 46 m . The entrance channel is 13 m long and 6 m high, then narrows to dimensions of 1 × 2 m and gives access to a main hall with dimensions of 12 × 12m . The cave has a relatively small number of speleothems ( Rnjak 2014 ) . Type specimens : Holotype and paratypes were mounted in HYDRO-Matrix© on glass slides and de- posited in the Croatian Biospeleological Society Collection under the accession number TAM1 ( holotype ) , TAM2 and TAM3 ( paratypes ). Differential diagnosis : Centropyxis bipilata sp. nov. resembles Centropyxis laevigata in general view. However, in C. laevigata the inner and the anterior wall of the shell are connected by a broad flange covered with thick amorphous scales and the test is larger (70–150 µm ( Penard, 1902 ). Schönborn et al. (1983) described a similar species from different locations in Germany as C. laevigata , but their species always had three struts. C. bipilata differs from Centropyxis malvin- ensis that has the same size and apertural border but two lateral flanges in the middle part ( Vucetich, 1975 ). It can also be distinguished from Centropyxis obscu- rus that doesn’t have a clear rim around the aperture nor any struts ( Chardez, 1990 ). It differs from Centropyxis delicatula that is smaller, has a variable number of 3–5 struts and no pronounced apertural rim ( Penard, 1902 ). Biogeography : The Dinaric karst area ( Mihevc et al., 2010 ) and isolated mountain karst area in the northern part of Croatia ( Lacković et al., 2011 ). Centropyxis bipilata sp. nov. has not been detected in the surface habitats close to the researched caves (data not shown within this research), even though surface habitats have not been systematically studied yet.