Vannella

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 : 65

Vannella

The vannellid strains belonged to two lineages in different parts of the Vannella ML phylogeny ( Fig. 1A). The first lineage had low bootstrap and moderate PP support and contained four Microcystis -associated strains interspersed between 3 Vannella simplex and 1 uncultured clone sequence (p -distances 0.3–3.5%, number of differing positions 4–63). Its sister taxon was Vannella persistens (2.0–3.6%, 24–68). The sec- ond lineage (upper part of the phylogeny) had moderate bootstrap and maximal PP support and consisted of six Microcystis -associated Vannella strains (p -distances 0.8–3.0%, 13–28 differing positions). Its closest rela- tives were unidentified Vannella strains or uncultured clones from natural samples (2.4–9.1%, 32–98).

The locomotive cells of the vannellid strains A17 WVB, A1 SMB, A6 HEB and A7 HEB, all closely related to V. simplex ( Fig. 1C), shared a mainly flabel- late to semicircular, rarely spatulate shape, a length/ width ratio of 0.4–1.6 (mean 0.8) and an average coverage with hyaloplasm about half of the cell length anteriorly, but differed in average size. Locomotive forms of strains A17 WVB, A1 SMB and A6 HEB /A7 HEB were respectively 38–79 (mean 49), 20–56 (35) and 15–60 (31) µm long and respectively 45–83 (65), 20–61 (43) and 20–85 (41) µm wide. On several occasions, tro- phozoites of strain A17 WVB were observed containing a flagellum-like pseudopodium, either very long and straight or contracted and helical in appearance ( Fig. 1C 17–19). The trophozoites of strain A17 WVB were always covered with fecal pellets. For the other 3 strains this feature disappeared after several months of cultivation, however fecal pellets of about 9 µm in diameter could always be observed freely dispersed in the culture medium.

Locomotive amoebae of strains A4P4 ZHB, A2 SMB, A2 FBB, A1 DVDPB, A2 DVDPB, A3 DVDPB, all belonging to the second vannellid lineage, were fan-shaped however with considerable variations. They could adopt a semicircular ( Figs 1B 1–2), flabellate ( Figs 1B 4–5) or spatulate shape containing a pronounced tail up to 21 µm long ( Fig. 1B 3, 8). Feeding amoeba attached on Microcystis colonies ( Fig. 1B 9) had a greatest linear dimension of 15–50 µm (mean 31). The hyaloplasm formed a broad margin around the cell and covered 13–71% (40%) of the cell anteriorly. When floating cells settled and became locomotive, the frontal hyaline area could display a wavy front edge ( Figs 1B 2, 4, 6) and sometimes contained longitudinal ridges ( Fig. 1B 7) as a result of transverse wave formation. The vesicular nucleus was 2–7 µm in diameter and contained a centrally located nucleolus of 1–5 µm in diameter (arrowheads in Figs 1B 1–4). Usually one small contractile vacuole was present posteriorly (black arrows in Figs 1B 1, 7, 8), with up to 3 vacuoles in some individuals. Floating amoebae were either radial shaped, 10–35 µm in diameter, symmetric, displaying radially 5–11 pointed and basally thickened (up to 6 µm in width) pseudopodia up to 50 µm in length, or some- times irregularly shaped with only one to a few thick and round-tipped pseudopodia ( Figs 1B 10–11) or in a few cases even without pseudopodia. Fecal pellets, varying in size between 5 and 13 µm, were present in the culture medium but did not tend to adhere posteriorly. Elliptical to ovoid cysts were observed a few times in old cultures. These had a thin wall (0.5 µm) and a di- ameter of 11–32 µm. Each had either a round opening of about 6 µm in diameter (white arrow in Fig. 1B 12) or a bone-shaped narrow slit of about 11 µm in length (white arrow in Fig. 1B 13).

Thirty Vannella species are currently recognized, 24 from marine and brackish, 4 from freshwater and 2 from soil environments ( Smirnov et al. 2007). Our phylogenetic analysis placed 4 of our vannellid strains in a clade containing several strains of Vannella simplex , a species with a wide geographically distribution, isolated from both freshwater and brackish habitats and genetically rather variable ( Smirnov et al. 2002, Nassonova et al. 2010). Since our strains also share the relatively large cell size and the coverage by fecal pellets, both typical for V. simplex isolates ( Smirnov et al. 2002), we believe they indeed belong to this species. Our remaining vannellid strains were much smaller in size, lacked the coverage by fecal pellets and formed a separate clade with uncultured clones or unidentified Vannella ’s as their closest relatives. They were clearly separated from two other freshwater species, V. miroides and V. lata . No sequence information is available for V. cirifera , the only other freshwater Vannella species known, however this species was announced a nomen dubium by Smirnov et al. (2002, 2007). For this reason and because of its specific ecology we declared the strains of our second lineage as belonging to a new species, Vannella planctonica .