Establishment of the new genus Parasincirra n. g.
Diagnosis. Amphisiellidae with elongate body. Three frontal cirri. Amphisiellid median cirral row about as long as adoral zone. One short frontoventral cirral row. Cirrus III/2 and transverse cirri present. One right and one left marginal row. Three dorsal kineties. Caudal cirri and buccal cirrus lacking.
Etymology. Composite of the Greek prefix para- (close to; related; deviating) and the posterior part (-sincirra) of the genus name Hemisincirra Hemberger, 1985. This indicates that Parasincirra has a cirral pattern similar to the Hemisincirra. This similarity should indicate a relationship to Hemisincirra, but whereas the new genus lacks a buccal cirrus Hemisincirra possesses one. Like Hemisincirra, Parasincirra has a feminine gender.
Type species. Parasincirra sinica n. sp.
Type locality: Sludge soil (36º3'N; 103º49'E) in Lanzhou, China.
Remarks. We do not assign Hemisincirra interrupta (Foissner, 1982) Foissner in Berger, 2001 and H. vermicularis Hemberger, 1985 to our new genus Parasincirra although both species also lack a buccal cirrus. The main reasons are that either the ontogenetic or the molecular information remains unknown.
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Description of Parasincirra sinica n. sp.
Diagnosis. Size in vivo 90–160 × 20–40 μm. Body slender, fusiform to worm-shaped, with pointed posterior end. Cortical granules about 0.5 μm across, colourless and grouped around dorsal ciliary organelles. Contractile vacuole located slightly ahead of mid-body. Two to six (mostly four) macronuclear nodules. Adoral zone composed of 14–19 membranelles. Amphisiellid median cirral row terminating behind cytostome level, composed of invariably four cirri. Three frontal and one parabuccal cirri; frontoventral row consistently with two cirri; two to four transverse cirri. One left and one right marginal row, composed of 34–52 and 34–53 cirri respectively. Three bipolar dorsal kineties. Soil habitat.
Type locality. Flood drain, Lanzhou (36º03'N; 103º49'E), China.
Etymology. The species-group name sinica means the species was first discovered in China.
Morphological description. The body colourless to greyish, non-contractile but highly flexible, and thus cell outline variable, i.e., sigmoidal or curved (Fig. 2d). Generally slender, almost fusiform to worm-shaped. Anterior end narrowly rounded and posterior end more or less tapered to form a pointed tail that is more flexible and contractile than the rest of the cell (Fig. 2a, d, g, h), and unrecognisable in protargol preparations (Fig. 2e, f, i, j). Dorsoventrally flattened up to 2:1. Cells were 90–160 × 20–40 μm in living cells (n=6), with an average of 120 × 30 μm in prepared cells, with a ratio of length to width of about 3.5:1–7.5:1 in vivo and on average 4:1 in protargol preparations. Cortical granules colourless, round, about 0.5 μm in diameter, around dorsal ciliary organelles, visible also in protargol preparations (Fig. 2c, j). One contractile vacuole measuring about 13 μm in diameter in diastole, positioned near left margin, contracting at intervals of 10 s (Fig. 2g, h). Cytoplasm often packed with numerous small lipid droplets. Usually four (2-6) macronuclear nodules arranged along mid-line, or slightly left of it, behind buccal vertex and one to three, on average two, micronuclei attached, or near to, macronuclear nodules. Macronuclear nodules ellipsoid to ellipsoid, about 9–19 × 4–10 μm in size (after protargol staining) (Fig. 2j). Micronuclei about 2.9 × 2.4 μm in size (after protargol staining). Locomotion mainly by slowly crawling on substrate and debris, sometimes jerking back and forth. When suspended, cells often swim continuously in circles.
Infraciliature as shown in Fig. 2 (b, e, f, i-l). Adoral zone of membranelles (AZM) shaped as in other amphisiellid species, terminates at 11–20% (average about 16%) of body length, comprising 14–19 membranelles. Cilia of distal membranelles about 13 μm long. Buccal cavity small, endoral and paroral bending strongly and optically intersecting with each other at their lower middle regions (Fig. 2b, e, i, k).
Most somatic cirri relatively fine with cilia about 12–16 μm long. Consistently three relatively stout frontal cirri in an almost transverse pseudo row immediately behind several distal adoral membranelles, cilia about 15 μm long. Amphisiellid median cirral row (ACR) short and consisting of four cirri; commences at about the level of the rightmost frontal cirrus (about 6% of body length), or slightly lower, terminates at about level of buccal vertex (about 21% of body length). Parabuccal cirrus (cirrus III/2) located at the level of the middle region of the paroral and endoral. Frontoventral row in region between parabuccal cirrus and ACR, consistently composed of two cirri; commences at about the level of the second cirrus in ACR (about 8% of body length) and terminates ahead of the third cirrus in ACR (about 10% of body length). Three, rarely two or four, slightly subterminal transverse cirri, cilia of which about 16 μm long. Consistently one left and one right marginal row, each with 34–52 and 34–53 cirri, respectively. Right marginal cirral row begins dorsolaterally at anterior end of cell while left marginal cirral row begins at level of posterior end of adoral zone, both of them terminate caudally, but are not confluent (Fig. 2b, e, i, k, l).
Three dorsal kineties arranged in Gonostomum-pattern, with cilia about 3 μm in length, composed of about 13, 15 and 15 dikinetids, respectively, and arrange in a gradient; that is, kinety 3 commences apically, kinety 2 starts slightly behind kinety 3, while kinety 1 starts slightly behind kinety 2. All of them terminate at the posterior body end (Fig. 2f, j).
Morphogenesis during binary fission
Stomatogenesis. Cortical morphogenesis in Parasincirra sinica n. sp. mainly occurs in two zones: an anterior field for the proter and a posterior field for the opisthe.
In the opisthe, the first evidence of stomatogenesis during cell division is the appearance of groups of basal bodies on the cell surface, that is, the opisthe’s oral primordium, which is located in the end of ACR, indicating that parental basal bodies are incorporated in the primordium (Fig. 3a). These groups subsequently merge by further proliferation of basal bodies forming a single anarchic field and then the new adoral membranelles organise posteriad (Figs. 3b, 5e). The anlage for the undulating membranes (anlage I) is formed to the right of the oral primordium (Figs. 3c, 5e). Later, the left frontal cirrus develops from the anterior end of the UM-anlage (Figs. 4a, 5n). During the later stages, the differentiation of membranelles is completed, forming the new oral structure for the opisthe. Subsequently, UM-anlage gives rise to the leftmost frontal cirrus, new endoral and paroral for the opisthe (Figs. 4a, b, 5n).
In the proter, several of the proximal membranelles dedifferentiate into sparsely distrusted basal bodies, then the basal bodies differentiate into membranelles (Fig. 3c-e). The parental undulating membranes dedifferentiate into UM-anlage, then the basic development of the UM-anlage follows a similar pattern to that in the opisthe (Figs. 3b-e, g, 4a, 5d, e, m).
Development of the frontoventral-transverse cirri. The development of the somatic ciliature begins with the formation of the frontoventral-transverse cirral anlagen (FVT-anlagen). At the beginning, FVT-anlagen appear as a small group of basal bodies (Fig. 3a). Apparently, the parental frontoventral cirri are disaggregated and joined in the formation of the FVT-anlagen. Later, five FVT-anlagen are formed to the right of the UM-anlage in the proter as primary primordia (Figs. 3b, 5d). Then, the FVT-anlagen fragment in the middle to form two sets of anlagen, one set for the proter and the other for the opisthe (Figs. 3d, e, 5h, i).
Subsequently, cirri segregate from anterior to posterior in the following manner: anlage I develops the frontal cirrus I/1 (leftmost frontal cirrus); anlage II produces the middle frontal cirrus; anlage III generates a parabuccal cirrus and the rightmost frontal cirrus; anlage IV contributes two cirri forming a short frontoventral cirral row; anlage V produces the posterior two cirri in ACR, and anlage VI forms the anterior two cirri in ACR; while anlagen IV to VI produce one transverse cirrus each (Figs. 4a, b, 5m, p). Finally, the new cirri move towards their final positions.
Development of marginal rows and dorsal kineties. Within every parental marginal row a few cirri near the anterior end, and a few others below the mid-body, differentiate to form two separate anlagen
The dorsal kineties develop by intrakinetal basal body proliferation, i.e. two anlagen develop in each parental row. Subsequently, the new marginal cirri/kineties develop and replace the old ones (Figs. 3d-h, 4a-c, 5g, h, j, m).
Division of nuclear apparatus. The nuclear apparatus divides in the usual way for hypotrichs hence no need to describe in detail (Figs. 3f, h, 4c, 5k).
SSU rRNA gene sequence and phylogenetic analyses
The sequence of the 18S rRNA gene of Parasincirra sinica n. sp. (GenBank accession number: MN472864) is 1731 bp long and has a G + C content of 45.70%. Phylogenetic trees inferred from the SSU rDNA sequences using two different methods (ML and BI) show similar topologies. Therefore, only the ML topology is shown, with nodal support from both methods (Fig. 7).
Molecular phylogenetic analyses result in a clade containing four polytomies represented by Parasincirra sinica n. sp., two Uroleptoides species and Parabistichella variabilis Jiang et al., 2013 with high support (83 % ML, 1.00 BI, Fig. 7). They also confirm the polyphyly of other amphisiellids including species belonging to the type genus Amphisiella Gourret & Roeser, 1888.