Hartmannella, Alexeieff, 1912

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 : 68-72

Hartmannella / Copromyxa

The 18S rDNA sequences of the two strains most closely related to Hartmannella / Copromyxa were highly divergent (p -distance 9.4%, 113 differing positions). In the ML phylogeny ( Fig. 4A) they were most closely related to and had a basal position in the lineage including Copromyxa protea , Copromyxa / Hartmannella cantabrigiensis and an unidentified strain (‘Tubuli- nida sp.’) (p -distances 9.7–15.2%, 117–197 differing positions).

The locomotive amoebae (mainly seen in starved cultures, see Figs 4B 1, 3, 7) of strain A2 JEPDK were monotactic with anteriorly a short-lived but well pronounced hyaline cap ( Figs 4B 7, 10) of about 3 µm long and 6 µm wide that became rapidly filled with granu- loplasm prior to the formation of a new lateral lob-like pseudopodium ( Figs 4B 2, 5, 6, 11). These alternating lateral sprouting lobes gave the locomotion a sort of eruptive-like appearance and with any change of direction transformed monopodial movement swiftly in a bipodial ( Fig. 4B 4) or multipodial (several irregularly located pseudopodia, Fig. 4B 6) state. Pseudopodia were up to 13 µm long and 5 µm wide at the base.At times an eruptive lobe could be seen rippling down on one side of the amoeba ( Figs 4B 8, 9). No differentiated uroi- dal structures were observed. Length of limax-shaped amoebae was 20–59 µm (mean 33 µm), width 7–17 µm (11) and length/width ratio 2–4.9 (3.1). The locomotion rate was estimated at 0.35–0.88 µm s –1. Length during non-oriented movement was 10–43 µm (24), width 6–25 µm (13) and length/width ratio 1.0–4.3 (1.9). One vesicular oval nucleus of 5–10 µm long and 3–8 µm wide was present with one central to slightly eccentrically placed globular nucleolus of about 2–3 µm in diameter ( Figs 4B 1, 5). Usually one small contractile vacuole posteriorly, rarely up to 3 vacuoles in very large individuals. The cytoplasm always contained 8–22 opaque crystals (e.g. arrow in Fig. 4B 1) with a length and width of respectively 1.1–7.2 µm (average 2.4) and 0.7–3.4 µm (1.7), and several smaller, refractive spheres. The crystals could be single or paired and of irregular, truncated bi-pyramidal, cylindrical, centrally inclined cube-like or plate-like (sometimes divided in 4 equal squares) shape. Spherical floating forms showed a high resemblance with those of Saccamoeba limax ( Page 1988, p. 65) . They were occasionally observed after re-inoculation and had a diameter of 15–20 µm and radially displayed 5–10 small hyaline lobes of about 3 µm in diameter ( Fig. 4B 15). Cysts were spheri- cal with a diameter of 9–20 µm, a cell wall of about 1 µm thickness and sometimes displayed an inner wall ( Fig. 4B 16). These double-walled cysts are similar to so-called sphaerocysts observed in Copromyxa protea which may be part of a (para) sexual cycle ( Brown et al. 2011). Empty cysts contained a round pore of about 5 µm in diameter (not shown).

Locomotive amoebae (mainly seen in starved cultures, Figs 4C 1, 6 –9) of strain A5 DVDPB were monotactic, displaying anteriorly a short-lived but well pronounced hyaline cap ( Fig. 4C 9) about 5 µm long and 5 µm wide that was rapidly filled with granuloplasm just before a new lateral pseudopodium was formed. With any change of direction, monopodial movement was however easily transformed into a bipodial ( Figs 4C 6, 9) and eventually a multipodial (several irregularly located pseudopodia) state due to these alternating sprouting (eruptive) lobes. Pseudopodia were up to 25 µm long and about 5 µm wide at their base, laterally harboring a few small, sprouting, short-living subpseudopodia ( Figs 4C 3, 11) of which one eventually became the new leading pseudopodium. Limax-shaped amoebae were 20–55 µm long (mean 38) and 8–25 µm wide (13) with a length/width ratio of 1.4–6.7 (3.1). Amoebae during non-oriented movement (e.g. when feeding on Microcystis cells) were more irregularly shaped, 15–25 µm in diameter, with radially 5–12 small blunted, round-tipped pseudopodia ( Figs 4C 3 –5,10– 12). No differentiated uroidal structures were observed apart of a few adhesive uroidal filaments occasionally (slightly visible in Fig. 4C 9). One vesicular oval nucleus of 4–8 µm long and 4–7 µm wide with one cen- trally to slightly eccentrically located globular nucleolus of about 3 µm in diameter (arrow in Fig. 4C 5) was present. The nucleolus may contain a highly refractive central pore-like area (lacuna). Usually one contractile vacuole of about 6 µm in diameter present posteriorly (e.g. Figs 4C 9–12). The cytoplasm contained 10–20 small, spherical to irregular shaped, dark-colored, refractive, single or paired crystals of about 0.5–1.6 µm (1.0) in diameter (e.g. Figs 4C 5, 7, 11). Floating forms were occasionally observed after re-inoculation. They were spherical to slightly elongated, with a diameter of 15–20 µm, radially displaying 5–12 small hyaline lobes with a maximal length of 15 µm ( Figs 4C 13–15), gen- erally resembling those of Nolandella hibernica ( Page 1980, Fig. 4). Spherical cysts about 20 µm in diameter were occasionally present in starved cultures.

Together with Saccamoeba , Glaeseria , Cashia and Copromyxella (for the last two, 18S rDNA sequences are not available), Hartmannella and Copromyxa belong to the family Hartmannellidae ( Smirnov et al. 2011) . Currently, Hartmannella contains only three species ( Smirnov et al. 2011). Two of these are marine – H. lobifera and H. vacuolata – and one is from freshwater, Hartmannella cantabrigiensis ( Smirnov 1996 /97, Anderson et al. 1997). Copromyxa contains only one species, C. protea , however there is ongoing discussion as to whether H. cantabrigiensis should be included in Copromyxa ( Brown et al. 2011, Smirnov et al. 2011). The trophozoites of our strains were characterized by their aquatic lifestyle and absence of fruiting-body formation in contrast to Copromyxella and Copromyxa protea that are terrestrial, dung-inhabiting, aggregative, fruiting amoebae (slime molds) ( Raper et al. 1978, Brown et al. 2011); their clearly visible and rather extended hyaline cap in contrast to Glaeseria , Cashia and Saccamoeba ( Page 1988) ; their absence of a differentiated uroidal structure in contrast to Saccamoeba ( Page 1988, Anderson et al. 1997, Corsaro et al. 2010); and their generally eruptive-like movement which is usually non-eruptive in all other members of the family, including Hartmannella ( Page 1988, Smirnov 1996 /97, Anderson et al. 1997). The last character is however not very reliable since Raper et al. (1978) observed potentially ‘explosive’ pseudopodia in trophozoites of all described Copromyxella species, Page (1980) mentioned uncharacteristic eruptive activity in some Nolandella species, Smirnov et al. (2011) observed occasional eruptions in a Hartmannella / Copromyxa strain and Watson et al. (2014) mentioned semi-eruptive movement in the recently established hartmannellid Ptolemeba .

Brown et al. (2011) emended the generic diagnosis of Copromyxa by also including aquatic, potentially non-fruiting limax-shaped amoebae ( H. cantabrigiensis , Hartmannella strain 4/3Da/10) similar in morphology to our strains. For this reason and because we also observed a double-walled cyst stage very similar to the sphaerocysts described for C. protea in one of our cultures, we placed our strains within the genus Copromyxa . The phylogenetic distance between our strains and the other taxa within this clade warranted the description of two new species (based on the large divergence in 18S rDNA sequence), C. microcystidis and C. vandevyveri .