Nephrococcus serbicus, a new coccoid cyanobacterial species from Božana Cave, Serbia Author Popović, Slađana University of Belgrade, Scientific Institution, Institute of Chemistry, Technology and Metallurgy, Department of Ecology and Technoeconomics, Belgrade, Serbia & University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden “ Jevremovac ”, Belgrade, Serbia Author Simić, Gordana Subakov University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden “ Jevremovac ”, Belgrade, Serbia Author Korać, Aleksandra University of Belgrade, Faculty of Biology, Center for Electron Microscopy, Studentski trg 16, Belgrade, Serbia Author Golić, Igor University of Belgrade, Faculty of Biology, Center for Electron Microscopy, Studentski trg 16, Belgrade, Serbia Author Komárek, Jiří Institute of Botany, Academy of Sciences of the Czech Republic, Dukelská 135, CZ- 37982 Třeboň, Czech Republic text Phytotaxa 2016 2016-12-22 289 2 135 146 http://dx.doi.org/10.11646/phytotaxa.289.2.3 journal article 10.11646/phytotaxa.289.2.3 1179-3163 13644452 Nephrococcus serbicus S. Popović, G. Subakov Simić & Komárek , sp. nov. ( Figs 2–5 ) The thallus (comprising macroscopic flat layers) is composed from more or less spherical colonies, usually containing (2) 4–16 (64) cells, and with ± firm outer envelopes. The colonies are mucilaginous, unstratified or slightly stratified, with colonial slime that is colourless. The cells are organized ± in a wreath-like formation or agglomerated in the centre. Spherical colonies up to 29 μm in diameter, with cells (usually packed together relatively densely) 1.5–6.5 μm in diameter. The cell colour varies from blue-green to olive-green or yellowish. Type locality :— SERBIA . Visočka Banja: Božana Cave, biofilm from the stone (limestone) substrate of the cave wall, left side, 5–6 m from the cave entrance, 43˚38΄12.50΄΄N, 19˚55΄25.21΄΄E, 723 m elev. Collected by S. Popović , summer 2014 . Type: Holotype T0008 , preserved in formalin, here illustrated as Fig. 5 , LM: Figs 2–4 , at the Department of Algology , Mycology and Lichenology , the Institute of Botany and Botanical Garden “Jevremovac”, ( BEOU ), the Faculty of Biology , University of Belgrade , Serbia .— Iconotype : Fig. 5 , photo Fig. 2 . FIGURE 2. Nephrococcus colonies from natural material: a = 2-celled stadium with yellow-brown cells and cell envelopes; b = oval, yellow-brown cells aggregated in the centre of the colony; c = yellowish, irregularly rounded cells nearly arranged in the form of a wreath; d = olive-green to yellowish, irregularly rounded cells nearly arranged in the form of a wreath; e = blue-green kidney-shaped cells arranged in a wreath formation; f = blue-green kidney-shaped cells arranged in a wreath formation, with wreaths placed opposite one another; g, h = probably resting cells. Scale bar: 10 μm. FIGURE 3. Nephrococcus colonies stained with toluidine blue (polysaccharides are stained purple): a, c = stadiums with four cells; b = stadiums with eight cells; d = cells aggregated in the centre of the colony; e, f = wreath formation. Description (LM and CLM):— Colonies that were spherical, multicellular—initially 2-to 4-celled ( Fig. 2a and Fig. 3a, c , respectively) and later on many-celled, probably up to 64 ( Fig. 2b , Fig. 3d )—were enveloped by a wide, mucilaginous and mostly unstratified or slightly stratified ( Fig. 4n ) colourless envelope with a firm surface. The presence of 1-celled stadiums has not been recorded, but they can evidently occur (at least in the form of possible resting cells ( Figs 2g , h , 5a )). The envelope is more stratified in younger colonies (e.g., a 2-celled stadium). A firm surface is evident in natural and coloured materials ( Figs 2 , 3 ). The most frequently occurring colonies contained 4, 8 and 16 cells. The colony size of the many-celled stadiums varies from 25–29 μm, and the envelope width from 4–9 μm. The cells are aggregated in the centre of the colony, sometimes just densely packed ( Fig. 2b , Fig. 3d ), but are often arranged in the form of a wreath ( Figs 2e, f , 4m ). Usually one wreath can be seen in the first plan, but at least two wreaths are probably formed and they are usually placed opposite one another ( Fig. 2f ). It is possible for more wreaths to be present. The shape of the cells varies from round, oval ( Fig. 2b ) or an irregular round shape ( Fig. 2c, d ) to a kidney shape ( Fig. 2e, f ), which is most common. The cell colour varies from blue-green ( Fig. 2e, f ), olive-green ( Fig. 2d ) to yellowish ( Fig. 2c ) and yellow-brown ( Fig. 2b ). The cell content is homogeneous or slightly granular. The cell envelopes are visible only in 2-celled stadiums ( Fig. 2a ), and later are not visible or not present. The cell length is 3–6.5 μm, with the cell width in the middle part being 1.5–4 μm. CLM ( Fig. 4 ) turned out to be a good technique for visualizing the 3D spatial organization of cells in the Nephrococcus colony, using propidium iodide that binds with nucleic acids. Without disturbing the structure, a series of thin optical sections of the sample-Optic 2D images obtained at different intervals: a–l-was made. Cells are arranged irregularly at the centre of the colony (a–l) or in the form of a wreath (m–n). Slight lamellation of the colony envelope is clearly evident in Fig. 4n . Drawings of Nephrococcus serbicus are shown in Fig. 5 . Etymology :—The epithet indicates that this species is found in Serbia . Ecology :—This aerophytic cyanobacterium was found in the subaerophytic, wet, stony (limestone) substrates of Božana Cave (near the entrance), at an elevation of 723 m , in western Serbia during the summer period of 2014. Measured environmental parameters from the sampling site and parameters from the biofilm containing Nephrococcus are shown in Table 1 . The sampling site was relatively close to the entrance and T, RH and LI were, in general, influenced by the external climatic conditions. The biofilm was moderately wet, with a lot more inorganic than organic matter ( Table 1 ). TABLE 1 . Measured parameters from sampling site and biofilm containing Nephrococcus .

Parameter	Value/appearance
T (˚C)	19.8 ± 0.09
RH (%)	79 ± 3
LI (Lux)	571 ± 5
4.27	35.07	6.68	58.23
Biofilm colour	Blackish, green with yellow parts

FIGURE 4. CLM of Nephrococcus showing the 3D spatial organization of cells in the Nephrococcus colony, with a series of thin optical sections of the sample (a–l). FIGURE 5. The drawings of Nephrococcus serbicus : a = probably the resting cell; b = 2-celled stadium; c = 4-celled stadium; d = 8-celled stadium; e = many-celled stadium; f = wreath formation. Scale bar: 10 μm. Associated taxa and additional information :—Other cyanobacterial taxa found in the biofilm containing Nephrococcus were Asterocapsa sp. H .-J. Chu (1952: 97–100 ; pl. 1, figs 1–3, pl. 2, figs 8, 9, 14), Aphanocapsa muscicola ( Meneghini 1843 ) Wille (1919: 39 ; pl. I, II) Aphanothece caldariorum P. G. Richter (1880: 192) , Chroococcus ercegovicii Komárek & Anagnostidis (1995: 17 ; figs 1, 3), Gloeocapsa alpina Nägeli in Rabenhorst (1865: 40) , Gloeocapsa violacea Kützing (1847: 25 ; pl. 36, fig. IX), Gloeocapsa spp. Kützing (1843: 173–175) , Gloeothece rupestris ( Lyngbye 1819: 207 ; pl. 69) Bornet in Wittrock & Nordstedt (1880: 339) , Gloeothece sp. Nägeli (1849: 57–60) , Leptolyngbya foveolarum (Gomont 1892: 164, pl. IV/4, fig. 16; Anagnostidis & Komárek 1988: 391 ) and Scytonema sp. C .Agardh ex É.Bornet & C.Flahault (1886: 85–115). Besides mosses, one unidentified green alga was also present. The first axis of the performed CCA ( Fig. 6 ) showed positive correlation with LI (r=0.6682) and negative correlation with T (r=−0.6151). The second CCA axis also showed a positive correlation with LI (r=0.5640) and a slightly negative correlation with RH (r=−0.3347). The eigenvalue of the first CCA axis was 0.7173 indicating that most of the ecological information was reflected on this axis, but a significant part of the variability was also explained by the second axis (eigenvalue 0.5514). According to CCA, the sampling site containing Nephrococcus had the highest LI and the lowest T recorded. The included supplementary variables, water content and content of organic/inorganic matter (expressed as mg/cm 2 ), also had the highest values at this sampling site. The content of Chl-a was also high. CCA showed that the sampling site that contained Nephrococcus was distinct from all the other sampling sites explored in Božana Cave, and many taxa that were not recorded at other sampling sites were documented at S8. FIGURE 6. CCA of cyanobacterial and algal taxa found in Božana Cave, with T, RH and LI as explanatory variables and sampling sites (S1–S8), chlorophyll-a (expressed as μg/cm 2 ), the water content and the content of organic/inorganic matter (expressed as mg/cm 2 ) as supplementary variables. The main difference points between all Nephrococcus species are shown in Table 2 . TABLE 2 . A brief description of all Nephrococcus species. Legend: * Referred to one small colony ** Data obtained or measured from the illustrations *** In developed colonies —Missing