Apsiktrata gracilis ( Penard, 1922 ) Foissner et al., 1994

Jiang, Limin, Wang, Congcong, Liao, Lijian, Al-Farraj, Saleh A., Warren, Alan & Hu, Xiaozhong, 2024, Re-evaluation of the systematic position of the order Prostomatida (Protista: Ciliophora), with the establishment of two new genera and two new species, Zoological Journal of the Linnean Society 200 (4), pp. 849-864 : 855-857

Apsiktrata gracilis ( Penard, 1922) Foissner et al., 1994

( Fig. 5 ; Table 2 )

Description of Qingdao population: Cell size in vivo ~40–70 μm × ~15–30 μm, on average 50 μm × 20 μm, with a length-to-width ratio of 2–3:1 ( Fig. 5A, F, I, J ). Body generally obovate, circular in apical view, anterior end broadly rounded, posterior half significantly narrowed; well-fed individuals more rounded in mid-region ( Fig. 5A, F, I, J ). Cytostome apical, circular in outline; oral basket funnel shaped, ~10–14 μm × ~2–4 μm, with slender fibres attached to anterior end; collar-like bulge around anterior end of body ( Fig. 5A, J, N ). Surface of body with shallow longitudinal furrows, along which somatic kineties run ( Fig. 5K ). Silverline system comprises irregular parallelograms connected by slanting fibrils to form a grid ( Fig. 5G, M ). Cytoplasm colourless, usually with several large food vacuoles coloured vividly blue–green by algal food and with several small brown or colourless globules ( Fig. 5I ). Extrusomes rod shaped, ~2–3 μm long after protargol staining, distributed along somatic kineties, lying orthogonal to pellicle ( Fig. 5E, H, K, L, Q ). One macronucleus, ellipsoidal, ~9–13 μm × ~8–11 μm after protargol staining, with several large spherical nucleoli, located slightly above equatorial region near cell mid-line; one micronucleus, globular, ~1 μm × ~1.5 μm after protargol staining, adjacent to macronucleus ( Fig. 5A, P ). Contractile vacuole in posterior third of body, ~11 μm in diameter when fully expanded ( Fig. 5A, F, J ). Locomotion by swimming slowly in upper layer of water, with an irregular trajectory.

Oral ciliature consisting only of a ring of radially arranged circumoral dikinetids, each kinetosome bearing a cilium ~5 μm long ( Fig. 5B, D, O ). Most somatic cilia ~8–10 μm long; single caudal cilium ~20–30 μm long ( Fig. 5A ). In total, 19–25 longitudinal somatic kineties composed of monokinetids. Kinetids uniformly spaced in anterior half of body, forming seven to nine regular transverse rings ( Fig. 5B–D ).

Molecular data and phylogenetic analyses

The SSU rRNA gene sequences of the three isolates obtained in the present study have been deposited in the GenBank database with the length (in base pairs),GC content and GenBank accession numbers as follows: Platina marina (1616 bp, 42.57%, OQ538389); Parametacystis pulchra (1715 bp, 39.65%, OQ538388); and Apsiktrata gracilis (1542 bp, 42.15%, OQ538387). The SSU rRNA gene sequence-based tree was constructed as shown in Figure 6 . Given that the topologies of the ML and BI trees were basically concordant, only the ML tree is presented, with support values from both algorithms indicated on the branches.

Our phylogenetic analyses revealed that the class Prostomatea was not monophyletic, owing to the class Plagiopylea nesting within it ( Fig. 6 ). The orders Prorodontida and Prostomatida were also non-monophyletic. The order Prorodontida was divided into four major clades: Placidae , Colepidae + Prorodontida e + Holophryidae, Urotrichida + Plagiocampidae + Cryptocaryon idae + LagynusidaeandBalanionidae + Hexasterias.Thepossibility of the order Prorodontida being monophyletic was rejected by the AU test (log likelihood: 1.41818184; AU value: 0.015).

Even with the newly obtained data from the present study, only five 18S rRNA gene sequences of prostomatids were available,and these were clustered in three distantly separated clades. Platina marina (OQ538389) and Parametacystis pulchra (OQ538388) clustered together with high support (ML 92%, BI 1.00) and then grouped with Foissnerophrys alveolata (MW481206) with full support (ML 100%, BI 1.00; Fig. 6 , marked with #). This clade nested deep within the class Prostomatea and was sister to the class Plagiopylea with maximal support (ML 100%, BI 1.00). Metacystis similis Zhang et al., 2015 (KT27408), the only available sequence of the genus Metacystis , clustered with species assigned to the families Holophryidae and Prorodontidae within the order Prorodontida with high support (ML 99%, BI 1.00) and occupied the basal position within this assemblage. Apsiktrata gracilis (OQ538387), the only species of the family Apsiktratidae , was sister to the family Placidae , although with no or low support (ML 48%, BI 0.92) The possibility of the order Prostomatida being monophyletic was rejected by the AU test (log likelihood: 1.41818184, AU value: 4 × 10 −16).

The putative secondary structures of the SSU rRNA gene V9 region of 18 species (16 prostomateans and two plagiopyleans) were predicted and compared ( Fig. 7A ). These taxa shared a similar secondary structure; the general model is formed by a long helix, with some median loops and a terminal loop. Species of the order Prostomatida shared the same structure as plagiopyleans, i.e. at a relatively conserved position near the 3ʹ end, they had a bulge consisting of two nucleotides (G and A) at bases 10 and 11. However, in the same position, Apsiktrata and other species of the order Prorodontida had a bulge consisting of three nucleotides (G, A and U) at bases 10, 11 and 12, respectively, and in some of these species there was also a bulge with two nucleotides (C and G) at bases 15 and 16, respectively.

The taxa that were the focus of the present study were divided into two groups, i.e. group 1 and group 2. The species of group 1 ( Prostomatida + Trimyemidae ) shared unique nucleotides at 31 sites in the SSU rRNA gene sequences ( Fig. 7B ). In contrast, the species of group 2 ( Apsiktratidae + some representative prorodontids) shared unique nucleotides at 40 sites ( Fig. 7B ).

Figures

Figure 5. Drawing (A–H) and photomicrographs (I–Q) of Qingdao population of Apsiktrata gracilis from life (A, E, F, H–L, N), after protargol staining (B–D, O–Q) and after silver nitrate staining (G, M). A, lateral view of a typical individual, showing general appearance of the body, circumoral cilia (arrowheads) and caudal cilium (arrow). B, C, side view, showing the somatic kineties and circumoral kinety (arrow). D, ciliary pattern of the anterior portion of the cell, showing the circumoral kinety (CK) and the collar on the oral basket (arrow). E, side view of cell, showing the distribution of rod-shaped extrusomes (arrows) in the cortex. F, shape variants. Arrowheads show the contractile vacuole. G, silverline system. H, distribution of extrusomes (arrowhead). I, lateral view of a typical cell, showing the body shape. J, collar on oral basket (arrowhead) and contractile vacuole (arrow). K, the distribution of kinetosomes (arrows) and extrusomes (arrowheads). L, distribution of the extrusomes (arrows). M, the silverline system. N, showing the shape of the oral basket.O, ciliary pattern of the anterior portion of the cell, showing the circumoral kinety (arrow). P, macronucleus and micronucleus (arrow). Q, showing the extrusomes (arrowheads). Scale bars: 25 μm.

Figure 6. Maximum likelihood (ML) tree inferred from small subunit ribosomal RNA (SSU rRNA) gene sequences, showing the phylogenetic positions of the three newly sequenced species (bold font). Numbers near the nodes represent the ML bootstrap values and Bayesian inference (BI) posterior probabilities, respectively. Fully supported (100/1.00) branches are marked with filled circles.Asterisks (*) indicate disagreements between the ML and BI trees.The scale bar corresponds to five substitutions per 100 nucleotide positions.

Figure 7. A, putative secondary structure of V9 region of small subunit ribosomal RNA (SSU rRNA) gene of 18 representative species. The major differences are indicated by shading. B, the unique sites of the alignment of SSU rRNA gene sequences for the two groups. Group 1 contains Prostomatida and Trimyemidae; group 2 contains Apsiktratidae and some representative prorodontids.