Korotnevella, Goodkov, 1988

Van Wichelen, Jeroen, D’Hondt, Sofie, Claeys, Myriam, Vyverman, Wim, Berney, Cédric, Bass, David & Vanormelingen, Pieter, 2016, A Hotspot of Amoebae Diversity: 8 New Naked Amoebae Associated with the Planktonic Bloom-forming Cyanobacterium Microcystis, Acta Protozoologica 55 (2), pp. 61-87 : 67-68

Korotnevella

All the dactylopodial strains were placed in the genus Korotnevella , which appeared monophyletic with good bootstrap and posterior probability support ( Fig. 2A). The Microcystis -associated Korotnevella strains fell into two lineages, containing two and five strains respectively, without representatives of known species. P -distances were 4.9–5.9% (75–99 differing positions) between both lineages and 0.0–1.7% (0–23) within a lineage.

Locomotive amoebae belonging to the first line- age, strains A4 DVDPB and A1 JEPDK, were triangular, elongated or irregularly shaped ( Fig. 2B). No differen- tiated uroidal structures were observed. Feeding amoebae (on Microcystis colonies) were spherical to cylindrical without dactylopodia ( Fig. 2B 7) but sometimes with a very long and thin sub-pseudopodium when maintained in starved conditions. Length of the locomotive form was 16–41 µm (mean 28), width 6–20 µm (11) and length/width ratio 1.2–6.0 (2.6). One vesicular globular nucleus about 2–3 µm in diameter was present containing one slightly eccentrically located globular nucleolus of about 1–2 µm, occasionally showing a highly refractive central pore-like area (lacuna) un- der phase-contrast illumination (arrow in Fig. 2B 7). Floating forms were spherical with a diameter of 10–16 µm (14) and displaying 4–10 long, hyaline, pointed pseudopodia of maximally 25 µm long. Large spheri- cal planktonic aggregates of up to several hundred individuals were observed occasionally in older cultures. Cysts were never observed. The scales, covering the entire cell surface, consisted of an elliptical disc with upwards curved edges and a centrally located cone-like spine giving it a ‘witches hat’ appearance ( Fig. 3a, c). The length of the scales observed at top view ( Fig. 3a) varied from 193–327 nm (277), the width from 124– 203 nm (172) and the L/W ratio from 1.2–2.1 (1.6). The spine length observed in cross-section ( Fig. 3c) was 47–97 nm (76), width at the base 56–124 nm (80) and L/W ratio 0.5–1.5 (1.0).

The locomotive amoebae from the second lineage, strains A8 WVB, A16 WVB, A21 WVB, A54 WVB and A1 LMS, were all triangular, elongated or irregularly shaped due to the formation of short to deeply inclined pseudopodia on one or both ends or all around the cell body ( Figs 2C 3, 4, 8 –12). No differentiated uroidal structures were observed. Amoebae during non-oriented movement were elongated ( Figs 2C 2, 6) or irregularly shaped ( Fig. 2C 1), containing several small, sometimes bifurcated, round-tipped pseudopodia. Feeding amoebae (on Microcystis colonies) were spherical to cylindrical without clear pseudopodia ( Fig. 2C 5). Starved amoebae had a similar cell shape but sometimes contained a very long and thin sub-pseudopodium. Cell length during locomotion was 20–65 µm (mean 37), width 6–32 µm (14) and the length/ width ratio 1.2–6.0 (2.8). One vesicular globular nucleus about 6 µm in diameter was present containing one slightly eccentrically located globular nucleolus of about 3 µm (arrows in Figs 2C 8, 10, 12), occasionally showing a highly refractive central pore-like area (lacuna) under phase-contrast illumination (faintly visible in Fig. 2C 8). Floating forms ( Fig. 2C 13) were of radial type, spherical, with a diameter of 10–25 µm (15) and displaying 1–20 (9) long, hyaline, pointed pseudopodia with a length of 20–100 µm (52). The entire cell surface was covered with scales, only visible with electron microscopy ( Figs 3b, d). In cross-section ( Fig. 3d), these were generally oriented in a roof-tile pattern (imbricated). The scales consisted of an elliptical disk with upwards curved edges and a centrally located cone-like spine giving it a ‘witches hat’ appearance. The length of the scales observed at top view was 260–391 nm (333), the width 153–206 nm (180) and the length/width ratio 1.5–2.6 (1.9). The spine length measured on scales observed in cross-section was 98–163 nm (121), the width measured at the base of the spine 61–131 nm (91) and the length/width ratio 0.8–2.2 (1.4). Starved amoebae tended to float and aggregate into large, spherical colo- nies of several tens to hundreds of individuals. A few times, a globular cyst-like body with a diameter of 15– 20 µm was observed in the cultures ( Fig. 2C 7) .

Korotnevella is a genus of scale-bearing amoebae all characterized by the absence of parasomes ( Smirnov et al. 2011). Currently 8 Korotnevella species are recognized – 4 from freshwater, 3 from marine habitats and 1 from terrestrial origin – that are mainly distinguished on the base of their scale morphology ( O’Kelly et al. 2001, Udalov 2015). The Korotnevella species without 18S rDNA sequence data are K. nivo , a marine species with crown-like scales ( Smirnov 1996 /97), 3 freshwater species, being K. bulla with basket-like scales ( Page 1981), K. diskophora with disc-like scales (Smirnov 1999) and K. limbata having both scale types ( Udalov 2015) and the terrestrial K. heteracantha also bearing both scale types ( Udalov 2015). TEM analysis of the Microcystis -associated Korotnevella strains from both lineages showed the presence of disc-like scales ( Fig. 3) very similar to the scales of K. diskophora . Apart from some small differences in scale dimensions and spine length, our strains mainly differed from K. diskophora , by having a spherical floating form with radially many pointed pseudopodia (irregular with few pseudopodia in K. diskophora ) and by their preference for Microcystis and thus planktonic occurrence (bacterivore and benthic lifestyle in K. diskophora ). Apart from the phylogenetic divergence, both Microcystis -associated Korotnevella lineages also differ in their morphology since the size of the trophozoites and of the scales and spines is smaller in strains of the first lineage (A4 DVDPB and A1 JEPDK). Based on the above-mentioned differences between both Microcystis -associated Korotnevella lineages and with K. diskophora , we described both lineages as new species, K. pelagolacustris and K. jeppesenii .